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Sample records for critical lysine residues

  1. Critical lysine residues of Klf4 required for protein stabilization and degradation

    SciTech Connect

    Lim, Key-Hwan; Kim, So-Ra; Ramakrishna, Suresh; Baek, Kwang-Hyun

    2014-01-24

    Highlights: • Klf4 undergoes the 26S proteasomal degradation by ubiquitination on its multiple lysine residues. • Essential Klf4 ubiquitination sites are accumulated between 190–263 amino acids. • A mutation of lysine at 232 on Klf4 elongates protein turnover. • Klf4 mutants dramatically suppress p53 expression both under normal and UV irradiated conditions. - Abstract: The transcription factor, Krüppel-like factor 4 (Klf4) plays a crucial role in generating induced pluripotent stem cells (iPSCs). As the ubiquitination and degradation of the Klf4 protein have been suggested to play an important role in its function, the identification of specific lysine sites that are responsible for protein degradation is of prime interest to improve protein stability and function. However, the molecular mechanism regulating proteasomal degradation of the Klf4 is poorly understood. In this study, both the analysis of Klf4 ubiquitination sites using several Klf4 deletion fragments and bioinformatics predictions showed that the lysine sites which are signaling for Klf4 protein degradation lie in its N-terminal domain (aa 1–296). The results also showed that Lys32, 52, 232, and 252 of Klf4 are responsible for the proteolysis of the Klf4 protein. These results suggest that Klf4 undergoes proteasomal degradation and that these lysine residues are critical for Klf4 ubiquitination.

  2. Folding simulations of alanine-based peptides with lysine residues.

    PubMed Central

    Sung, S S

    1995-01-01

    The folding of short alanine-based peptides with different numbers of lysine residues is simulated at constant temperature (274 K) using the rigid-element Monte Carlo method. The solvent-referenced potential has prevented the multiple-minima problem in helix folding. From various initial structures, the peptides with three lysine residues fold into helix-dominated conformations with the calculated average helicity in the range of 60-80%. The peptide with six lysine residues shows only 8-14% helicity. These results agree well with experimental observations. The intramolecular electrostatic interaction of the charged lysine side chains and their electrostatic hydration destabilize the helical conformations of the peptide with six lysine residues, whereas these effects on the peptides with three lysine residues are small. The simulations provide insight into the helix-folding mechanism, including the beta-bend intermediate in helix initiation, the (i, i + 3) hydrogen bonds, the asymmetrical helix propagation, and the asymmetrical helicities in the N- and C-terminal regions. These findings are consistent with previous studies. PMID:7756550

  3. Accessibility and mobility of lysine residues in. beta. -lactoglobulin

    SciTech Connect

    Brown, E.M.; Pfeffer, P.E.; Kumosinski, T.F.; Greenberg, R.

    1988-07-26

    N/sup epsilon/-(/sup 2/H/sub 6/)Isopropyllysyl-..beta..-lactoglobulin was prepared by reductive alkylation of ..beta..-lactoglobulin with (/sup 2/H/sub 6/)acetone and NaBH/sub 4/ to provide a /sup 2/H (NMR) probe for the study of lysine involvement in lipid-protein interactions. Amino acid analysis showed 80% of the protein's 15 lysine residues to be labeled. Unmodified lysine residues were located through peptide maps produced from CNBr, tryptic, and chymotryptic digests of the labeled protein. Average correlation times calculated from /sup 2/H NMR spectra were 20 and 320 ps for 8.7 and 3.3 residues, respectively, in 6 M guanidine hydrochloride; in nondenaturing solution, values of 70 and 320 ps were obtained for 6.5 and 3.2 residues, respectively, with the remaining 2.3 modified residues not observed, suggesting that side chains of lysine residues in unordered or flexible regions were more mobile than those in stable periodic structures. /sup 2/H NMR spectra of the protein complexed with dipalmitoylphosphatidylcholine confirmed the extrinsic membrane protein type behavior of ..beta..-lactoglobulin previously reported from /sup 31/P NMR studies of the phospholipids complexed with ..beta..-lactoglobulin. Although no physiological function has yet been identified, comparison of these results with the X-ray structure supports the hypothesis that residues not accessible for modification may help to stabilize the cone-shaped ..beta..-barrel thought to contain binding sites for small lipid-soluble molecules.

  4. On methylene-bridged cysteine and lysine residues in proteins.

    PubMed

    Ruszkowski, Milosz; Dauter, Zbigniew

    2016-09-01

    Cysteine residues ubiquitously stabilize tertiary and quaternary protein structure by formation of disulfide bridges. Here we investigate another linking interaction that involves sulfhydryl groups of cysteines, namely intra- and intermolecular methylene-bridges between cysteine and lysine residues. A number of crystal structures possessing such a linkage were identified in the Protein Data Bank. Inspection of the electron density maps and re-refinement of the nominated structures unequivocally confirmed the presence of Lys-CH2 -Cys bonds in several cases. PMID:27261771

  5. Selective Deletion of the Internal Lysine Residue from the Peptide Sequence by Collisional Activation

    NASA Astrophysics Data System (ADS)

    Banerjee, Shibdas; Mazumdar, Shyamalava

    2012-11-01

    The gas-phase peptide ion fragmentation chemistry is always the center of attraction in proteomics to analyze the amino acid sequence of peptides and proteins. In this work, we describe the formation of an anomalous fragment ion, which corresponds to the selective deletion of the internal lysine residue from a series of lysine containing peptides upon collisional activation in the ion trap. We detected several water-loss fragment ions and the maximum number of water molecules lost from a particular fragment ion was equal to the number of lysine residues in that fragment. As a consequence of this water-loss phenomenon, internal lysine residues were found to be deleted from the peptide ion. The N,N-dimethylation of all the amine functional groups of the peptide stopped the internal lysine deletion reaction, but selective N-terminal α-amino acetylation had no effect on this process indicating involvement of the side chains of the lysine residues. The detailed mechanism of the lysine deletion was investigated by multistage CID of the modified and unmodified peptides, by isotope labeling and by energy resolved CID studies. The results suggest that the lysine deletion might occur through a unimolecular multistep mechanism involving a seven-membered cyclic imine intermediate formed by the loss of water from a lysine residue in the protonated peptide. This intermediate subsequently undergoes degradation reaction to deplete the interior imine ring from the peptide backbone leading to the deletion of an internal lysine residue.

  6. Glycated Lysine Residues: A Marker for Non-Enzymatic Protein Glycation in Age-Related Diseases

    PubMed Central

    Ansari, Nadeem A.; Moinuddin; Ali, Rashid

    2011-01-01

    Nonenzymatic glycosylation or glycation of macromolecules, especially proteins leading to their oxidation, play an important role in diseases. Glycation of proteins primarily results in the formation of an early stage and stable Amadori-lysine product which undergo further irreversible chemical reactions to form advanced glycation endproducts (AGEs). This review focuses these products in lysine rich proteins such as collagen and human serum albumin for their role in aging and age-related diseases. Antigenic characteristics of glycated lysine residues in proteins together with the presence of serum autoantibodies to the glycated lysine products and lysine-rich proteins in diabetes and arthritis patients indicates that these modified lysine residues may be a novel biomarker for protein glycation in aging and age-related diseases. PMID:21725160

  7. Charge Stabilization and Entropy Reduction of Central Lysine Residues in

    SciTech Connect

    St-Jean, M.; Blonski, C; Sygusch, J

    2009-01-01

    Fructose-1,6-bisphosphate muscle aldolase is an essential glycolytic enzyme that catalyzes reversible carbon-carbon bond formation by cleaving fructose 1,6-bisphosphate to yield dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde phosphate. To elucidate the mechanistic role of conserved amino acid Asp-33, Asn-33 and Ser-33 mutants were examined by kinetic and structural analyses. The mutations significantly compromised enzymatic activity and carbanion oxidation in presence of DHAP. Detailed structural analysis demonstrated that, like native crystals, Asp-33 mutant crystals, soaked in DHAP solutions, trapped Schiff base-derived intermediates covalently attached to Lys-229. The mutant structures, however, exhibited an abridged conformational change with the helical region (34-65) flanking the active site as well as pK{sub a} reductions and increased side chain disorder by central lysine residues, Lys-107 and Lys-146. These changes directly affect their interaction with the C-terminal Tyr-363, consistent with the absence of active site binding by the C-terminal region in the presence of phosphate. Lys-146 pKa reduction and side chain disorder would further compromise charge stabilization during C-C bond cleavage and proton transfer during enamine formation. These mechanistic impediments explain diminished catalytic activity and a reduced level of carbanion oxidation and are consistent with rate-determining proton transfer observed in the Asn-33 mutant. Asp-33 reduces the entropic cost and augments the enthalpic gain during catalysis by rigidifying Lys-107 and Lys-146, stabilizing their protonated forms, and promoting a conformational change triggered by substrate or obligate product binding, which lower kinetic barriers in C-C bond cleavage and Schiff base-enamine interconversion.

  8. ERAD of proteins containing aberrant transmembrane domains requires ubiquitylation of cytoplasmic lysine residues

    PubMed Central

    Briant, Kit; Koay, Yee-Hui; Otsuka, Yuka; Swanton, Eileithyia

    2015-01-01

    ABSTRACT Clearance of misfolded proteins from the endoplasmic reticulum (ER) is mediated by the ubiquitin-proteasome system in a process known as ER-associated degradation (ERAD). The mechanisms through which proteins containing aberrant transmembrane domains are degraded by ERAD are poorly understood. To address this question, we generated model ERAD substrates based on CD8 with either a non-native transmembrane domain but a folded ER luminal domain (CD8TMD*), or the native transmembrane domain but a misfolded luminal domain (CD8LUM*). Although both chimeras were degraded by ERAD, we found that the location of the folding defect determined the initial site of ubiquitylation. Ubiquitylation of cytoplasmic lysine residues was required for the extraction of CD8TMD* from the ER membrane during ERAD, whereas CD8LUM* continued to be degraded in the absence of cytoplasmic lysine residues. Cytoplasmic lysine residues were also required for degradation of an additional ERAD substrate containing an unassembled transmembrane domain and when a non-native transmembrane domain was introduced into CD8LUM*. Our results suggest that proteins with defective transmembrane domains are removed from the ER through a specific ERAD mechanism that depends upon ubiquitylation of cytoplasmic lysine residues. PMID:26446255

  9. Regulation of translesion DNA synthesis: posttranslational modification of lysine residues in key proteins

    PubMed Central

    McIntyre, Justyna; Woodgate, Roger

    2015-01-01

    Posttranslational modification of proteins often controls various aspects of their cellular function. Indeed, over the past decade or so, it has been discovered that posttranslational modification of lysine residues plays a major role in regulating translesion DNA synthesis (TLS) and perhaps the most appreciated lysine modification is that of ubiquitination. Much of the recent interest in ubiquitination stems from the fact that proliferating cell nuclear antigen (PCNA) was previously shown to be specifically ubiquitinated at K164 and that such ubiquitination plays a key role in regulating TLS. In addition, TLS polymerases themselves are now known to be ubiquitinated. In the case of human polymerase η, ubiquitination at four lysine residues in its C-terminus appears to regulate its ability to interact with PCNA and modulate TLS. Within the past few years, advances in global proteomic research has revealed that many proteins involved in TLS are, in fact, subject to a previously underappreciated number of lysine modifications. In this review, we will summarize the known lysine modifications of several key proteins involved in TLS; PCNA and Y-family polymerases η, ι, κ and Rev1 and we will discuss the potential regulatory effects of such modification in controlling TLS in vivo. PMID:25743599

  10. Proline and lysine residues provide modulatory switches in amyloid formation: Insights from prion protein.

    PubMed

    Kraus, Allison

    2016-01-01

    Amyloidogenic proteins have an increased propensity to reorganize into the highly structured, β sheet rich structures that characterize amyloid. The probability of attaining these highly structured assemblies is influenced by multiple factors, including amino acid composition and environmental conditions. Evolutionary selection for amino acid sequences that prevent amyloid formation could further modulate amyloid-forming propensity. Indeed, we have recently identified specific proline and lysine residues, contained within a highly conserved central region of prion protein (PrP), that impede PrP amyloid formation in vitro. These prolines are mutated in certain forms of the human familial genetic disease, Gerstmann-Straüssler-Schneiker (GSS) syndrome. Here, I discuss the influence of these proline and lysine residues on PrP amyloid formation and how such anti-amyloidogenic primary amino acid sequences might be modulated to influence protein amyloidogenicity. PMID:26864641

  11. Cardiolipin binds selectively but transiently to conserved lysine residues in the rotor of metazoan ATP synthases.

    PubMed

    Duncan, Anna L; Robinson, Alan J; Walker, John E

    2016-08-01

    The anionic lipid cardiolipin is an essential component of active ATP synthases. In metazoans, their rotors contain a ring of eight c-subunits consisting of inner and outer circles of N- and C-terminal α-helices, respectively. The beginning of the C-terminal α-helix contains a strictly conserved and fully trimethylated lysine residue in the lipid head-group region of the membrane. Larger rings of known structure, from c9-c15 in eubacteria and chloroplasts, conserve either a lysine or an arginine residue in the equivalent position. In computer simulations of hydrated membranes containing trimethylated or unmethylated bovine c8-rings and bacterial c10- or c11-rings, the head-groups of cardiolipin molecules became associated selectively with these modified and unmodified lysine residues and with adjacent polar amino acids and with a second conserved lysine on the opposite side of the membrane, whereas phosphatidyl lipids were attracted little to these sites. However, the residence times of cardiolipin molecules with the ring were brief and sufficient for the rotor to turn only a fraction of a degree in the active enzyme. With the demethylated c8-ring and with c10- and c11-rings, the density of bound cardiolipin molecules at this site increased, but residence times were not changed greatly. These highly specific but brief interactions with the rotating c-ring are consistent with functional roles for cardiolipin in stabilizing and lubricating the rotor, and, by interacting with the enzyme at the inlet and exit of the transmembrane proton channel, in participation in proton translocation through the membrane domain of the enzyme. PMID:27382158

  12. Cardiolipin binds selectively but transiently to conserved lysine residues in the rotor of metazoan ATP synthases

    PubMed Central

    Duncan, Anna L.

    2016-01-01

    The anionic lipid cardiolipin is an essential component of active ATP synthases. In metazoans, their rotors contain a ring of eight c-subunits consisting of inner and outer circles of N- and C-terminal α-helices, respectively. The beginning of the C-terminal α-helix contains a strictly conserved and fully trimethylated lysine residue in the lipid head-group region of the membrane. Larger rings of known structure, from c9-c15 in eubacteria and chloroplasts, conserve either a lysine or an arginine residue in the equivalent position. In computer simulations of hydrated membranes containing trimethylated or unmethylated bovine c8-rings and bacterial c10- or c11-rings, the head-groups of cardiolipin molecules became associated selectively with these modified and unmodified lysine residues and with adjacent polar amino acids and with a second conserved lysine on the opposite side of the membrane, whereas phosphatidyl lipids were attracted little to these sites. However, the residence times of cardiolipin molecules with the ring were brief and sufficient for the rotor to turn only a fraction of a degree in the active enzyme. With the demethylated c8-ring and with c10- and c11-rings, the density of bound cardiolipin molecules at this site increased, but residence times were not changed greatly. These highly specific but brief interactions with the rotating c-ring are consistent with functional roles for cardiolipin in stabilizing and lubricating the rotor, and, by interacting with the enzyme at the inlet and exit of the transmembrane proton channel, in participation in proton translocation through the membrane domain of the enzyme. PMID:27382158

  13. Lysyl Hydroxylase 3 Modifies Lysine Residues to Facilitate Oligomerization of Mannan-Binding Lectin

    PubMed Central

    Risteli, Maija; Ruotsalainen, Heli; Bergmann, Ulrich; Venkatraman Girija, Umakhanth; Wallis, Russell; Myllylä, Raili

    2014-01-01

    Lysyl hydroxylase 3 (LH3) is a multifunctional protein with lysyl hydroxylase, galactosyltransferase and glucosyltransferase activities. The LH3 has been shown to modify the lysine residues both in collagens and also in some collagenous proteins. In this study we show for the first time that LH3 is essential for catalyzing formation of the glucosylgalactosylhydroxylysines of mannan-binding lectin (MBL), the first component of the lectin pathway of complement activation. Furthermore, loss of the terminal glucose units on the derivatized lysine residues in mouse embryonic fibroblasts lacking the LH3 protein leads to defective disulphide bonding and oligomerization of rat MBL-A, with a decrease in the proportion of the larger functional MBL oligomers. The oligomerization could be completely restored with the full length LH3 or the amino-terminal fragment of LH3 that possesses the glycosyltransferase activities. Our results confirm that LH3 is the only enzyme capable of glucosylating the galactosylhydroxylysine residues in proteins with a collagenous domain. In mice lacking the lysyl hydroxylase activity of LH3, but with untouched galactosyltransferase and glucosyltransferase activities, reduced circulating MBL-A levels were observed. Oligomerization was normal, however and residual lysyl hydroxylation was compensated in part by other lysyl hydroxylase isoenzymes. Our data suggest that LH3 is commonly involved in biosynthesis of collagenous proteins and the glucosylation of galactosylhydroxylysines residues by LH3 is crucial for the formation of the functional high-molecular weight MBL oligomers. PMID:25419660

  14. The emerging characterization of lysine residue deacetylation on the modulation of mitochondrial function and cardiovascular biology

    PubMed Central

    Lu, Zhongping; Scott, Iain; Webster, Bradley R.; Sack, Michael N.

    2009-01-01

    There is emerging recognition of a novel fuel and redox sensing regulatory program that controls cellular adaptation via non-histone protein lysine-residue acetyl post-translation modifications. This program functions in tissues with high energy demand and oxidative capacity and is highly enriched in the heart. Deacetylation is regulated by NAD+-dependent activation of the sirtuin family of proteins while acetyltransferase modifications are controlled by less clearly delineated acetyltransferases. Subcellular localization specific protein targets of lysine-acetyl modification have been identified in the nucleus, cytoplasm and mitochondria. Despite distinct subcellular localizations, these modifications appear, in large part, to modify mitochondrial properties including respiration, energy production, apoptosis and anti-oxidant defenses. These mitochondrial regulatory programs are important in cardiovascular biology, although how protein acetyl modifications effects cardiovascular pathophysiology has not been extensively explored. This review will introduce the role of non-histone protein lysine-residue acetyl modifications, discuss their regulation and biochemistry and present the direct and indirect data implicating their involvement in the heart and vasculature. PMID:19850949

  15. Identification of lysine 346 as a functionally important residue for pyridoxal 5'-phosphate binding and catalysis in lysine 2, 3-aminomutase from Bacillus subtilis.

    PubMed

    Chen, D; Frey, P A

    2001-01-16

    Lysine 2,3-aminomutase (LAM) catalyzes the interconversion of L-lysine and L-beta-lysine. The enzyme contains pyridoxal 5'-phosphate (PLP) and a [4Fe-4S] center and requires S-adenosylmethionine (SAM) for activity. The hydrogen transfer is mediated by the 5'-deoxyadenosyl radical generated in a reaction of the iron-sulfur cluster with SAM. PLP facilitates the radical rearrangement by forming a lysine-PLP aldimine, in which the imine group participates in the isomerization mechanism. We here report the identification of lysine 346 as important for PLP binding and catalysis. Reduction of LAM with NaBH(4) rapidly inactivated the enzyme with concomitant UV/visible spectrum changes characteristic of reduction of an aldimine formed between PLP and lysine. Following reduction with NaBH(4) and proteolysis with trypsin, a single phosphopyridoxyl peptide of 36 amino acid residues was identified by reverse-phase liquid chromatography/mass spectrometry (LC/MS). The purified phosphopyridoxyl peptide exhibited an absorption band at 325 nm, and its identity was further confirmed by tandem mass spectrometry (MS/MS) sequencing. The bound PLP is linked to lysine 346 in a PGGGGK (PLP) structure. The sequence of this binding motif is conserved in LAMs from Bacillus and Clostridium and other homologous proteins but is distinct from the PLP-binding motifs found in other PLP enzymes. The function of lysine 346 was further studied by site-directed mutagenesis. The purified K346Q mutant was inactive, and its content of PLP was only approximately 15% of that of the wild-type enzyme. The data indicate that the formation of the aldimine linkage between lysine 346 and PLP is important for LAM catalysis. Sequences similar to the PLP-binding motifs in other enzymes were also present in LAM. However, lysine residues within these motifs neither are the PLP-binding sites in LAM nor are directly involved in LAM catalysis. This study represents the first comprehensive investigation of PLP binding in

  16. Identification of family determining residues in Jumonji-C lysine demethylases: A sequence-based, family wide classification.

    PubMed

    Slama, Patrick

    2016-03-01

    Histone post-translational modifications play a critical role in the regulation of gene expression. Methylation of lysines at N-terminal tails of histones has been shown to be involved in such regulation. While this modification was long considered to be irreversible, two different classes of enzymes capable of carrying out the demethylation of histone lysines were recently identified: the oxidases, such as LSD1, and the oxygenases (JmjC-containing). Here, a family-wide analysis of the second of these classes is proposed, with over 300 proteins studied at the sequence level. We show that a correlated evolution analysis yields some position/residue pairs which are critical at comparing JmjC sequences and enables the classification of JmjC domains into five families. A few positions appear more frequently among conditions, such as positions 23 (directly C-terminal to the second iron ligand), 24, 252 and 253 (directly N-terminal to a conserved Asn). Implications of family conditions are studied in detail on PHF2, revealing the meaningfulness of the sequence-derived conditions at the structural level. These results should help obtain insights on the diversity of JmjC-containing proteins solely by considering some of the amino acids present in their JmjC domain. PMID:26757344

  17. Superoxide reduction by a superoxide reductase lacking the highly conserved lysine residue

    DOE PAGESBeta

    Teixeira, Miguel; Cabelli, Diane; Pinto, Ana F.; Romao, Celia V.; Pinto, Liliana C.; Huber, Harald; Saraiva, Ligia M.; Todorovic, Smilja

    2014-12-05

    Superoxide reductases (SORs) are the most recently identified superoxide detoxification systems, being found in microorganisms from the three domains of life. These enzymes are characterized by a catalytic mononuclear iron site, with one cysteine and four histidine ligands of the ferrous active form. A lysine residue in the –EKHVP– motif, located close to the active site, has been considered to be essential for the enzyme function, by contributing to the positive surface patch that attracts the superoxide anion and by controlling the chemistry of the catalytic mechanism through a hydrogen bond network. However, we show here that this residue ismore » substituted by non-equivalent amino acids in several putative SORs from Archaea and unicellular Eukarya. In this work, we focus on mechanistic and spectroscopic studies of one of these less common enzymes, the SOR from the hyperthermophilic crenarchaeon Ignicoccus hospitalis. We employ pulse radiolysis fast kinetics and spectroscopic approaches to study the wild-type enzyme (₋E₂₃T₂₄HVP₋), and two mutants, T24K and E23A, the later mimicking enzymes lacking both the lysine and glutamate (a ferric ion ligand) of the motif. The efficiency of the wild type protein and mutants in reducing superoxide is comparable to other SORs, revealing the robustness of these enzymes to single mutations.« less

  18. Mapping of anion binding sites on cytochrome c by differential chemical modification of lysine residues.

    PubMed Central

    Osheroff, N; Brautigan, D L; Margoliash, E

    1980-01-01

    The carbonate binding site on horse cytochrome c was mapped by comparing the yields of carboxydinitrophenyl-cytochromes c, each with a single carboxydinitrophenyl-substituted lysine residue per molecule, when the modification reaction was carried out in the presence and absence of carbonate. The site is located on the "left surface" of the protein and consists of lysine residues 72 and/or 73 as well as 86 and/or 87 (Carbonate Site). Although one of the binding sites for phosphate on cytochrome c (Phosphat Site I) is located near the carbonate site, the sites are distinctly different since carbonate does not displace bound phosphate, as monitored by 31P NMR. Furthermore, citrate interacts with Phosphate Site I with high affinity, whereas chloride, acetate, borate, and cacodylate have a much lower affinity for this site, if they bind to it at all. The affinity of phosphate for Phosphate Site I (KD = 2 X 10(-4) M) is at least 1 order of magnitude higher than it is for other sites of interaction. Images PMID:6254024

  19. Methylation of RNA polymerase II non-consensus Lysine residues marks early transcription in mammalian cells

    PubMed Central

    Dias, João D; Rito, Tiago; Torlai Triglia, Elena; Kukalev, Alexander; Ferrai, Carmelo; Chotalia, Mita; Brookes, Emily; Kimura, Hiroshi; Pombo, Ana

    2015-01-01

    Dynamic post-translational modification of RNA polymerase II (RNAPII) coordinates the co-transcriptional recruitment of enzymatic complexes that regulate chromatin states and processing of nascent RNA. Extensive phosphorylation of serine residues at the largest RNAPII subunit occurs at its structurally-disordered C-terminal domain (CTD), which is composed of multiple heptapeptide repeats with consensus sequence Y1-S2-P3-T4-S5-P6-S7. Serine-5 and Serine-7 phosphorylation mark transcription initiation, whereas Serine-2 phosphorylation coincides with productive elongation. In vertebrates, the CTD has eight non-canonical substitutions of Serine-7 into Lysine-7, which can be acetylated (K7ac). Here, we describe mono- and di-methylation of CTD Lysine-7 residues (K7me1 and K7me2). K7me1 and K7me2 are observed during the earliest transcription stages and precede or accompany Serine-5 and Serine-7 phosphorylation. In contrast, K7ac is associated with RNAPII elongation, Serine-2 phosphorylation and mRNA expression. We identify an unexpected balance between RNAPII K7 methylation and acetylation at gene promoters, which fine-tunes gene expression levels. DOI: http://dx.doi.org/10.7554/eLife.11215.001 PMID:26687004

  20. Superoxide reduction by a superoxide reductase lacking the highly conserved lysine residue

    SciTech Connect

    Teixeira, Miguel; Cabelli, Diane; Pinto, Ana F.; Romao, Celia V.; Pinto, Liliana C.; Huber, Harald; Saraiva, Ligia M.; Todorovic, Smilja

    2014-12-05

    Superoxide reductases (SORs) are the most recently identified superoxide detoxification systems, being found in microorganisms from the three domains of life. These enzymes are characterized by a catalytic mononuclear iron site, with one cysteine and four histidine ligands of the ferrous active form. A lysine residue in the –EKHVP– motif, located close to the active site, has been considered to be essential for the enzyme function, by contributing to the positive surface patch that attracts the superoxide anion and by controlling the chemistry of the catalytic mechanism through a hydrogen bond network. However, we show here that this residue is substituted by non-equivalent amino acids in several putative SORs from Archaea and unicellular Eukarya. In this work, we focus on mechanistic and spectroscopic studies of one of these less common enzymes, the SOR from the hyperthermophilic crenarchaeon Ignicoccus hospitalis. We employ pulse radiolysis fast kinetics and spectroscopic approaches to study the wild-type enzyme (₋E₂₃T₂₄HVP₋), and two mutants, T24K and E23A, the later mimicking enzymes lacking both the lysine and glutamate (a ferric ion ligand) of the motif. The efficiency of the wild type protein and mutants in reducing superoxide is comparable to other SORs, revealing the robustness of these enzymes to single mutations.

  1. Large shifts in pKa values of lysine residues buried inside a protein

    PubMed Central

    Isom, Daniel G.; Castañeda, Carlos A.; Cannon, Brian R.; García-Moreno E., Bertrand

    2011-01-01

    Internal ionizable groups in proteins are relatively rare but they are essential for catalysis and energy transduction. To examine molecular determinants of their unusual and functionally important properties, we engineered 25 variants of staphylococcal nuclease with lysine residues at internal positions. Nineteen of the Lys residues have depressed pKa values, some as low as 5.3, and 20 titrate without triggering any detectable conformational reorganization. Apparently, simply by being buried in the protein interior, these Lys residues acquired pKa values comparable to those of naturally occurring internal ionizable groups involved in catalysis and biological H+ transport. The pKa values of some of the internal Lys residues were affected by interactions with surface carboxylic groups. The apparent polarizability reported by the pKa values varied significantly from location to location inside the protein. These data will enable an unprecedented examination of the positional dependence of the dielectric response of a protein. This study also shows that the ability of proteins to withstand the presence of charges in their hydrophobic interior is a fundamental property inherent to all stable proteins, not a specialized adaptation unique to proteins that evolved to depend on internal charges for function. PMID:21389271

  2. Large shifts in pKa values of lysine residues buried inside a protein.

    PubMed

    Isom, Daniel G; Castañeda, Carlos A; Cannon, Brian R; García-Moreno, Bertrand

    2011-03-29

    Internal ionizable groups in proteins are relatively rare but they are essential for catalysis and energy transduction. To examine molecular determinants of their unusual and functionally important properties, we engineered 25 variants of staphylococcal nuclease with lysine residues at internal positions. Nineteen of the Lys residues have depressed pK(a) values, some as low as 5.3, and 20 titrate without triggering any detectable conformational reorganization. Apparently, simply by being buried in the protein interior, these Lys residues acquired pK(a) values comparable to those of naturally occurring internal ionizable groups involved in catalysis and biological H(+) transport. The pK(a) values of some of the internal Lys residues were affected by interactions with surface carboxylic groups. The apparent polarizability reported by the pK(a) values varied significantly from location to location inside the protein. These data will enable an unprecedented examination of the positional dependence of the dielectric response of a protein. This study also shows that the ability of proteins to withstand the presence of charges in their hydrophobic interior is a fundamental property inherent to all stable proteins, not a specialized adaptation unique to proteins that evolved to depend on internal charges for function. PMID:21389271

  3. N(ε)-Carboxymethyl Modification of Lysine Residues in Pathogenic Prion Isoforms.

    PubMed

    Choi, Yeong-Gon; Shin, Hae-Young; Kim, Jae-Il; Choi, Eun-Kyoung; Carp, Richard I; Kim, Yong-Sun

    2016-07-01

    The most prominent hallmark of prion diseases is prion protein conversion and the subsequent deposition of the altered prions, PrP(Sc), at the pathological sites of affected individuals, particularly in the brain. A previous study has demonstrated that the N-terminus of the pathogenic prion isoform (PrP(Sc)) is modified with advanced glycation end products (AGEs), most likely at one or more of the three Lys residues (positions 23, 24, and 27) in the N-terminus (23KKRPKP28). The current study investigated whether N(ε)-(carboxymethyl)lysine (CML), a major AGE form specific to Lys residues produced by nonenzymatic glycation, is an AGE adduct of the N-terminus of PrP(Sc). We show that CML is linked to at least one Lys residue at the N-terminus of PrP(Sc) in 263K prion-infected hamster brains and at least one of the eight Lys residues (positions 101, 104, 106, 110, 185, 194, 204, and 220) in the proteinase K (PK)-resistant core region of PrP(Sc). The nonenzymatic glycation of the Lys residue(s) of PrP(Sc) with CML likely occurs in the widespread prion-deposit areas within infected brains, particularly in some of the numerous tyrosine hydroxylase-positive thalamic and hypothalamic nuclei. CML glycation does not occur in PrP(C) but is seen in the pathologic PrP(Sc) isoform. Furthermore, the modification of PrP(Sc) with CML may be closely involved in prion propagation and deposition in pathological brain areas. PMID:25983034

  4. Identification of the active-site lysine residues of two biosynthetic 3-dehydroquinases.

    PubMed Central

    Chaudhuri, S; Duncan, K; Graham, L D; Coggins, J R

    1991-01-01

    The lysine residues involved in Schiff-base formation at the active sites of both the 3-dehydroquinase component of the pentafunctional arom enzyme of Neurospora crassa and of the monofunctional 3-dehydroquinase of Escherichia coli were labelled by treatment with 3-dehydroquinate in the presence of NaB3H4. Radioactive peptides were isolated by h.p.l.c. following digestion with CNBr (and in one case after further digestion with trypsin). The sequence established for the N. crassa peptide was ALQHGDVVKLVVGAR, and that for the E. coli peptide was QSFDADIPKIA. An amended nucleotide sequence for the E. coli gene (aroD) that encode 3-dehydroquinase is also presented, along with a revised alignment of the deduced amino acid sequences for the biosynthetic enzymes. PMID:1826831

  5. Role of a helix B lysine residue in the photoactive site in channelrhodopsins.

    PubMed

    Li, Hai; Govorunova, Elena G; Sineshchekov, Oleg A; Spudich, John L

    2014-04-15

    In most studied microbial rhodopsins two conserved carboxylic acid residues (the homologs of Asp-85 and Asp-212 in bacteriorhodopsin) and an arginine residue (the homolog of Arg-82) form a complex counterion to the protonated retinylidene Schiff base, and neutralization of the negatively charged carboxylates causes red shifts of the absorption maximum. In contrast, the corresponding neutralizing mutations in some relatively low-efficiency channelrhodopsins (ChRs) result in blue shifts. These ChRs do not contain a lysine residue in the second helix, conserved in higher efficiency ChRs (Lys-132 in the crystallized ChR chimera). By action spectroscopy of photoinduced channel currents in HEK293 cells and absorption spectroscopy of detergent-purified pigments, we found that in tested ChRs the Lys-132 homolog controls the direction of spectral shifts in the mutants of the photoactive site carboxylic acid residues. Analysis of double mutants shows that red spectral shifts occur when this Lys is present, whether naturally or by mutagenesis, and blue shifts occur when it is replaced with a neutral residue. A neutralizing mutation of the Lys-132 homolog alone caused a red spectral shift in high-efficiency ChRs, whereas its introduction into low-efficiency ChR1 from Chlamydomonas augustae (CaChR1) caused a blue shift. Taking into account that the effective charge of the carboxylic acid residues is a key factor in microbial rhodopsin spectral tuning, these findings suggest that the Lys-132 homolog modulates their pKa values. On the other hand, mutation of the Arg-82 homolog that fulfills this role in bacteriorhodopsin caused minimal spectral changes in the tested ChRs. Titration revealed that the pKa of the Asp-85 homolog in CaChR1 lies in the alkaline region unlike in most studied microbial rhodopsins, but is substantially decreased by introduction of a Lys-132 homolog or neutralizing mutation of the Asp-212 homolog. In the three ChRs tested the Lys-132 homolog also alters

  6. Determination of lysine residues affinity labeled in the active site of yeast RNA polymerase II(B) by mutagenesis.

    PubMed Central

    Treich, I; Carles, C; Sentenac, A; Riva, M

    1992-01-01

    In a previous study, yeast RNA polymerase II(B) was affinity labeled with two nucleotide derivatives (III and VIII) (1). In both cases, the labeled site was localized to the C-terminal part of the B150 subunit. The potential target lysyl residues of derivative III were mapped to the conserved domain H, between Asn946 and Met999. In the present work, we have mutagenized to arginine the five lysines present in domain H. Three lysines can be replaced, individually or simultaneously, without affecting cell growth, and each mutated enzyme can still be affinity labeled. Hence one or both of the other two lysyl residues, Lys979 and Lys987, is the target of the affinity reagent. These two lysines were each found to be essential for cell viability. Derivative VIII labeled another domain in addition to domain H. Supported by analogous results obtained for E. coli RNA polymerase using derivative VIII (2), we hypothesized that the second domain labeled by this derivative in the B150 subunit was domain I. Mutagenesis of the unique lysine present in domain I demonstrated that Lys 1102 was the target of derivative VIII. These results indicate that in both prokaryotic and eukaryotic RNA polymerases, domains H and I are in close proximity and participate to the active site. Images PMID:1408783

  7. Human METTL20 Methylates Lysine Residues Adjacent to the Recognition Loop of the Electron Transfer Flavoprotein in Mitochondria*

    PubMed Central

    Rhein, Virginie F.; Carroll, Joe; He, Jiuya; Ding, Shujing; Fearnley, Ian M.; Walker, John E.

    2014-01-01

    In mammalian mitochondria, protein methylation is a relatively uncommon post-transcriptional modification, and the extent of the mitochondrial protein methylome, the modifying methyltransferases, and their substrates have been little studied. As shown here, the β-subunit of the electron transfer flavoprotein (ETF) is one such methylated protein. The ETF is a heterodimer of α- and β-subunits. Lysine residues 199 and 202 of mature ETFβ are almost completely trimethylated in bovine heart mitochondria, whereas ETFα is not methylated. The enzyme responsible for the modifications was identified as methyltransferase-like protein 20 (METTL20). In human 143B cells, the methylation of ETFβ is less extensive and is diminished further by suppression of METTL20. Tagged METTL20 expressed in HEK293T cells specifically associates with the ETF and promotes the trimethylation of ETFβ lysine residues 199 and 202. ETF serves as a mobile electron carrier linking dehydrogenases involved in fatty acid oxidation and one-carbon metabolism to the membrane-associated ubiquinone pool. The methylated residues in ETFβ are immediately adjacent to a protein loop that recognizes and binds to the dehydrogenases. Suppression of trimethylation of ETFβ in mouse C2C12 cells oxidizing palmitate as an energy source reduced the consumption of oxygen by the cells. These experiments suggest that the oxidation of fatty acids in mitochondria and the passage of electrons via the ETF may be controlled by modulating the protein-protein interactions between the reduced dehydrogenases and the β-subunit of the ETF by trimethylation of lysine residues. METTL20 is the first lysine methyltransferase to be found to be associated with mitochondria. PMID:25023281

  8. Human METTL20 methylates lysine residues adjacent to the recognition loop of the electron transfer flavoprotein in mitochondria.

    PubMed

    Rhein, Virginie F; Carroll, Joe; He, Jiuya; Ding, Shujing; Fearnley, Ian M; Walker, John E

    2014-08-29

    In mammalian mitochondria, protein methylation is a relatively uncommon post-transcriptional modification, and the extent of the mitochondrial protein methylome, the modifying methyltransferases, and their substrates have been little studied. As shown here, the β-subunit of the electron transfer flavoprotein (ETF) is one such methylated protein. The ETF is a heterodimer of α- and β-subunits. Lysine residues 199 and 202 of mature ETFβ are almost completely trimethylated in bovine heart mitochondria, whereas ETFα is not methylated. The enzyme responsible for the modifications was identified as methyltransferase-like protein 20 (METTL20). In human 143B cells, the methylation of ETFβ is less extensive and is diminished further by suppression of METTL20. Tagged METTL20 expressed in HEK293T cells specifically associates with the ETF and promotes the trimethylation of ETFβ lysine residues 199 and 202. ETF serves as a mobile electron carrier linking dehydrogenases involved in fatty acid oxidation and one-carbon metabolism to the membrane-associated ubiquinone pool. The methylated residues in ETFβ are immediately adjacent to a protein loop that recognizes and binds to the dehydrogenases. Suppression of trimethylation of ETFβ in mouse C2C12 cells oxidizing palmitate as an energy source reduced the consumption of oxygen by the cells. These experiments suggest that the oxidation of fatty acids in mitochondria and the passage of electrons via the ETF may be controlled by modulating the protein-protein interactions between the reduced dehydrogenases and the β-subunit of the ETF by trimethylation of lysine residues. METTL20 is the first lysine methyltransferase to be found to be associated with mitochondria. PMID:25023281

  9. Role of surface lysine residues of adipocyte fatty acid-binding protein in fatty acid transfer to phospholipid vesicles.

    PubMed

    Liou, H L; Storch, J

    2001-05-29

    The tertiary structure of murine adipocyte fatty acid-binding protein (AFABP) is a flattened 10-stranded beta-barrel capped by a helix-turn-helix segment. This helical domain is hypothesized to behave as a "lid" or portal for ligand entry into and exit from the binding cavity. Previously, we demonstrated that anthroyloxy-labeled fatty acid (AOFA) transfer from AFABP to phospholipid membranes occurs by a collisional process, in which ionic interactions between positively charged lysine residues on the protein surface and negatively charged phospholipid headgroups are involved. In the present study, the role of specific lysine residues located in the portal and other regions of AFABP was directly examined using site-directed mutagenesis. The results showed that isoleucine replacement for lysine in the portal region, including the alphaI- and alphaII-helices and the beta C-D turn, resulted in much slower 2-(9-anthroyloxy)palmitate (2AP) transfer rates to acidic membranes than those of native AFABP. An additive effect was found for mutant K22,59I, displaying the slowest rates of FA transfer. Rates of 2AP transfer from "nonportal" mutants on the beta-G and I strands were affected only moderately; however, a lysine --> isoleucine mutation in the nonportal beta-A strand decreased the 2AP transfer rate. These studies suggest that lysines in the helical cap domain are important for governing ionic interactions between AFABP and membranes. Furthermore, it appears that more than one distinct region, including the alphaI-helix, alphaII-helix, beta C-D turn, and the beta-A strand, is involved in these charge-charge interactions. PMID:11371211

  10. Nitrogen dioxide reaction with proteins: Evidence for peptide bond cleavage at lysine residues

    SciTech Connect

    Hood, D.B.

    1991-01-01

    Nitrogen dioxide (NO{sub 2}), an air pollutant produced by burning fossil fuels and a component of cigarette smoke, is thought to contribute to the pathogenesis of pulmonary diseases, such as emphysema. To gain information on the mechanism by which NO{sub 2} damages the lung, in vitro exposures of {alpha}{sub 1}-proteinase inhibitor ({alpha}{sub 1}-PI), elastin, bovine serum albumin (BSA), human serum albumin (HSA) and synthetic poly-L-lysine were performed. A genetic deficiency of {alpha}{sup 1}-PI predisposes humans to emphysema and NO{sub 2} has been hypothesized to damage {alpha}{sub 1}-PI, which would leave proteases such as human neutrophil elastase, (HNE) free to attack lung structural proteins. The ability of {alpha}{sub 1}-PI to inhibit HNE declined with exposure to 50% of the control value at molar ratios of NO{sub 2}:{alpha}{sub 1}-PI of 400:1 and greater. Exposure of {alpha}{sub 1}-PI to NO{sub 2} resulted in a 50% lose of immunoreactivity with either monoclonal or polyclonal antibodies in an enzyme-linked immunosorbent assay at molar ratios of NO{sub 2}:{alpha}{sub 1}-PI of essentially 100:1 and greater. The mechanisms of these effects were investigated via ultraviolet-visible spectroscopy and amino acid analysis. The remaining target molecules were labeled by reductive methylation of amino groups with {sup 3}H-HCHO prior to treatment with NO{sub 2} in aqueous solutions at physiological pH. Time course exposure of 5 mg {sup 3}H-insoluble bovine ligamentum nuchae elastin suspensions with up to 120 {mu}moles of NO{sub 2} resulted in 90% solubilization of the label. Amino acid analysis of the soluble and insoluble fractions from these exposures confirmed that 80% of the {sup 3}H-dimethyllysine residues were in the soluble fraction.

  11. Adaptation of the behaviour of an aspartic proteinase inhibitor by relocation of a lysine residue by one helical turn.

    PubMed

    Winterburn, Tim J; Wyatt, David M; Phylip, Lowri H; Berry, Colin; Bur, Daniel; Kay, John

    2006-08-01

    In addition to self-inhibition of aspartic proteinase zymogens by their intrinsic proparts, the activity of certain members of this enzyme family can be modulated through active-site occupation by extrinsic polypeptides such as the small IA3 protein from Saccharomyces cerevisiae. The unprecedented mechanism by which IA3 helicates to inhibit its sole target aspartic proteinase locates an i, i+4 pair of charged residues (Lys18+Asp22) on an otherwise-hydrophobic face of the amphipathic helix. The nature of these residues is not crucial for effective inhibition, but re-location of the lysine residue by one turn (+4 residues) in the helical IA3 positions its side chain in the mutant IA3-proteinase complex in an orientation essentially identical to that of the key lysine residue in zymogen proparts. The binding of the extrinsic mutant IA3 shows pH dependence reminiscent of that required for the release of intrinsic zymogen proparts so that activation can occur. PMID:16895485

  12. CPLM: a database of protein lysine modifications

    PubMed Central

    Liu, Zexian; Wang, Yongbo; Gao, Tianshun; Pan, Zhicheng; Cheng, Han; Yang, Qing; Cheng, Zhongyi; Guo, Anyuan; Ren, Jian; Xue, Yu

    2014-01-01

    We reported an integrated database of Compendium of Protein Lysine Modifications (CPLM; http://cplm.biocuckoo.org) for protein lysine modifications (PLMs), which occur at active ε-amino groups of specific lysine residues in proteins and are critical for orchestrating various biological processes. The CPLM database was updated from our previously developed database of Compendium of Protein Lysine Acetylation (CPLA), which contained 7151 lysine acetylation sites in 3311 proteins. Here, we manually collected experimentally identified substrates and sites for 12 types of PLMs, including acetylation, ubiquitination, sumoylation, methylation, butyrylation, crotonylation, glycation, malonylation, phosphoglycerylation, propionylation, succinylation and pupylation. In total, the CPLM database contained 203 972 modification events on 189 919 modified lysines in 45 748 proteins for 122 species. With the dataset, we totally identified 76 types of co-occurrences of various PLMs on the same lysine residues, and the most abundant PLM crosstalk is between acetylation and ubiquitination. Up to 53.5% of acetylation and 33.1% of ubiquitination events co-occur at 10 746 lysine sites. Thus, the various PLM crosstalks suggested that a considerable proportion of lysines were competitively and dynamically regulated in a complicated manner. Taken together, the CPLM database can serve as a useful resource for further research of PLMs. PMID:24214993

  13. Inhibitors of enzymes catalyzing modifications to histone lysine residues: structure, function and activity.

    PubMed

    Lillico, Ryan; Stesco, Nicholas; Khorshid Amhad, Tina; Cortes, Claudia; Namaka, Mike P; Lakowski, Ted M

    2016-05-01

    Gene expression is partly controlled by epigenetic mechanisms including histone-modifying enzymes. Some diseases are caused by changes in gene expression that can be mitigated by inhibiting histone-modifying enzymes. This review covers the enzyme inhibitors targeting histone lysine modifications. We summarize the enzymatic mechanisms of histone lysine acetylation, deacetylation, methylation and demethylation and discuss the biochemical roles of these modifications in gene expression and in disease. We discuss inhibitors of lysine acetylation, deacetylation, methylation and demethylation defining their structure-activity relationships and their potential mechanisms. We show that there are potentially indiscriminant off-target effects on gene expression even with the use of selective epigenetic enzyme inhibitors. PMID:27173004

  14. The juxtamembrane lysine and arginine residues of surfactant protein C precursor influence palmitoylation via effects on trafficking.

    PubMed

    ten Brinke, A; Batenburg, J J; Gadella, B M; Haagsman, H P; Vaandrager, A B; van Golde, L M

    2001-08-01

    Surfactant protein (SP)-C propeptide (proSP-C) becomes palmitoylated on cysteines 5 and 6 before mature SP-C is formed by several proteolytic steps. To study the structural requirements for the palmitoylation of proSP-C, his-tagged human proSP-C (his-proSP-C) and his-proSP-C mutants were expressed in Chinese hamster ovary cells and analyzed by metabolic labeling with [(3)H]palmitate and immunocytochemistry. Substitution of cysteines 5 and 6 by serines showed that these were the only two cysteine residues palmitoylated in his-proSP-C. Substitution of the juxtamembrane basic residues lysine and arginine by uncharged glutamines led to a large decrease in palmitoylation level of proSP-C. The addition of brefeldin A nearly abolished this decrease for the lysine and double mutant; the palmitoylation of the arginine mutant increased also, but not to wild-type (WT) levels. Fluorescence immunocytochemistry showed that WT proSP-C was localized in punctate vesicles throughout the cell, whereas the mutant lacking the juxtamembrane positive charges was found more perinuclear, probably in the endoplasmic reticulum (ER). This indicates that the two basic juxtamembrane residues influence palmitoylation of proSP-C by preventing the transport of proSP-C out of the ER, implying that proSP-C becomes palmitoylated normally in a compartment distal to the ER. PMID:11509324

  15. Elicitin-Induced Distal Systemic Resistance in Plants is Mediated Through the Protein-Protein Interactions Influenced by Selected Lysine Residues.

    PubMed

    Uhlíková, Hana; Obořil, Michal; Klempová, Jitka; Šedo, Ondrej; Zdráhal, Zbyněk; Kašparovský, Tomáš; Skládal, Petr; Lochman, Jan

    2016-01-01

    Elicitins are a family of small proteins with sterol-binding activity that are secreted by Phytophthora and Pythium sp. classified as oomycete PAMPs. Although α- and β-elicitins bind with the same affinity to one high affinity binding site on the plasma membrane, β-elicitins (possessing 6-7 lysine residues) are generally 50- to 100-fold more active at inducing distal HR and systemic resistance than the α-isoforms (with only 1-3 lysine residues). To examine the role of lysine residues in elicitin biological activity, we employed site-directed mutagenesis to prepare a series of β-elicitin cryptogein variants with mutations on specific lysine residues. In contrast to direct infiltration of protein into leaves, application to the stem revealed a rough correlation between protein's charge and biological activity, resulting in protection against Phytophthora parasitica. A detailed analysis of proteins' movement in plants showed no substantial differences in distribution through phloem indicating differences in consequent apoplastic or symplastic transport. In this process, an important role of homodimer formation together with the ability to form a heterodimer with potential partner represented by endogenous plants LTPs is suggested. Our work demonstrates a key role of selected lysine residues in these interactions and stresses the importance of processes preceding elicitin recognition responsible for induction of distal systemic resistance. PMID:26904041

  16. Elicitin-Induced Distal Systemic Resistance in Plants is Mediated Through the Protein–Protein Interactions Influenced by Selected Lysine Residues

    PubMed Central

    Uhlíková, Hana; Obořil, Michal; Klempová, Jitka; Šedo, Ondrej; Zdráhal, Zbyněk; Kašparovský, Tomáš; Skládal, Petr; Lochman, Jan

    2016-01-01

    Elicitins are a family of small proteins with sterol-binding activity that are secreted by Phytophthora and Pythium sp. classified as oomycete PAMPs. Although α- and β-elicitins bind with the same affinity to one high affinity binding site on the plasma membrane, β-elicitins (possessing 6–7 lysine residues) are generally 50- to 100-fold more active at inducing distal HR and systemic resistance than the α-isoforms (with only 1–3 lysine residues). To examine the role of lysine residues in elicitin biological activity, we employed site-directed mutagenesis to prepare a series of β-elicitin cryptogein variants with mutations on specific lysine residues. In contrast to direct infiltration of protein into leaves, application to the stem revealed a rough correlation between protein’s charge and biological activity, resulting in protection against Phytophthora parasitica. A detailed analysis of proteins’ movement in plants showed no substantial differences in distribution through phloem indicating differences in consequent apoplastic or symplastic transport. In this process, an important role of homodimer formation together with the ability to form a heterodimer with potential partner represented by endogenous plants LTPs is suggested. Our work demonstrates a key role of selected lysine residues in these interactions and stresses the importance of processes preceding elicitin recognition responsible for induction of distal systemic resistance. PMID:26904041

  17. Fructose-1,6-bisphosphate aldolase of Neisseria meningitidis binds human plasminogen via its C-terminal lysine residue.

    PubMed

    Shams, Fariza; Oldfield, Neil J; Lai, Si Kei; Tunio, Sarfraz A; Wooldridge, Karl G; Turner, David P J

    2016-04-01

    Neisseria meningitidis is a leading cause of fatal sepsis and meningitis worldwide. As for commensal species of human neisseriae, N. meningitidis inhabits the human nasopharynx and asymptomatic colonization is ubiquitous. Only rarely does the organism invade and survive in the bloodstream leading to disease. Moonlighting proteins perform two or more autonomous, often dissimilar, functions using a single polypeptide chain. They have been increasingly reported on the surface of both prokaryotic and eukaryotic organisms and shown to interact with a variety of host ligands. In some organisms moonlighting proteins perform virulence-related functions, and they may play a role in the pathogenesis of N. meningitidis. Fructose-1,6-bisphosphate aldolase (FBA) was previously shown to be surface-exposed in meningococci and involved in adhesion to host cells. In this study, FBA was shown to be present on the surface of both pathogenic and commensal neisseriae, and surface localization and anchoring was demonstrated to be independent of aldolase activity. Importantly, meningococcal FBA was found to bind to human glu-plasminogen in a dose-dependent manner. Site-directed mutagenesis demonstrated that the C-terminal lysine residue of FBA was required for this interaction, whereas subterminal lysine residues were not involved. PMID:26732512

  18. V-type nerve agents phosphonylate ubiquitin at biologically relevant lysine residues and induce intramolecular cyclization by an isopeptide bond.

    PubMed

    Schmidt, Christian; Breyer, Felicitas; Blum, Marc-Michael; Thiermann, Horst; Worek, Franz; John, Harald

    2014-08-01

    Toxic organophosphorus compounds (e.g., pesticides and nerve agents) are known to react with nucleophilic side chains of different amino acids (phosphylation), thus forming adducts with endogenous proteins. Most often binding to serine, tyrosine, or threonine residues is described as being of relevance for toxicological effects (e.g., acetylcholinesterase and neuropathy target esterase) or as biomarkers for post-exposure analysis (verification, e.g., albumin and butyrylcholinesterase). Accordingly, identification of novel protein targets might be beneficial for a better understanding of the toxicology of these compounds, revealing new bioanalytical verification tools, and improving knowledge on chemical reactivity. In the present study, we investigated the reaction of ubiquitin (Ub) with the V-type nerve agents Chinese VX, Russian VX, and VX in vitro. Ub is a ubiquitous protein with a mass of 8564.8 Da present in the extra- and intracellular space that plays an important physiological role in several essential processes (e.g., proteasomal degradation, DNA repair, protein turnover, and endocytosis). Reaction products were analyzed by matrix-assisted laser desorption/ionization-time-of-flight- mass spectrometry (MALDI-TOF MS) and μ-high-performance liquid chromatography online coupled to UV-detection and electrospray ionization MS (μHPLC-UV/ESI MS). Our results originally document that a complex mixture of at least mono-, di, and triphosphonylated Ub adducts was produced. Surprisingly, peptide mass fingerprint analysis in combination with MALDI and ESI MS/MS revealed that phosphonylation occurred with high selectivity in at least 6 of 7 surface-exposed lysine residues that are essential for the biological function of Ub. These reaction products were found not to age. In addition, we herein report for the first time that phosphonylation induced intramolecular cyclization by formation of an isopeptide bond between the ε-amino group of a formerly phosphonylated

  19. Surface lysine residues modulate the collisional transfer of fatty acid from adipocyte fatty acid binding protein to membranes.

    PubMed

    Herr, F M; Matarese, V; Bernlohr, D A; Storch, J

    1995-09-19

    The transfer of unesterified fatty acids (FA) from adipocyte fatty acid binding protein (A-FABP) to phospholipid membranes is proposed to occur via a collisional mechanism involving transient ionic and hydrophobic interactions [Wootan & Storch (1994) J. Biol. Chem. 269, 10517-10523]. In particular, it was suggested that membrane acidic phospholipids might specifically interact with basic residues on the surface of A-FABP. Here we addressed whether lysine residues on the surface of the protein are involved in this collisional transfer mechanism. Recombinant A-FABP was acetylated to neutralize all positively charged surface lysine residues. Protein fluorescence, CD spectra, and chemical denaturant data indicate that acetylation did not substantially alter the conformational integrity of the protein, and nearly identical affinities were obtained for binding of the fluorescently labeled FA [12-(9-anthroyloxy)oleate] to native and acetylated protein. Transfer of 2-(9-anthroyloxy)palmitate (2AP) from acetylated A-FABP to small unilamellar vesicles (SUV) was 35-fold slower than from native protein. In addition, whereas the 2AP transfer rate from native A-FABP was directly dependent on SUV concentration, 2AP transfer from acetylated protein was independent on the concentration of acceptor membranes. Factors which alter aqueous-phase solubility of FA, such as ionic strength and acyl chain length and saturation, affected the AOFA transfer rate from acetylated but not native A-FABP. Finally, an increase in the negative charge density of the acceptor SUV resulted in a marked increase in the rate of transfer from native A-FABP but did not increase the rate from acetylated A-FABP.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7547918

  20. Essential lysine residues within transmembrane helix 1 of diphtheria toxin facilitate COPI binding and catalytic domain entry

    PubMed Central

    Trujillo, Carolina; Taylor-Parker, Julian; Harrison, Robert; Murphy, John R.

    2014-01-01

    The translocation of the diphtheria toxin catalytic domain from the lumen of early endosomes into the cytosol of eukaryotic cells is an essential step in the intoxication process. We have previously shown that the in vitro translocation of the catalytic domain from the lumen of toxin pre-loaded endosomal vesicles to the external medium requires the addition of cytosolic proteins including coatomer protein complex I (COPI) to the reaction mixture. Further, we have shown that transmembrane helix 1 plays an essential, but as yet undefined role in the entry process. We have used both site-directed mutagenesis and a COPI complex precipitation assay to demonstrate that interaction(s) between at least three lysine residues in transmembrane helix 1 are essential for both COPI complex binding and the delivery of the catalytic domain into the target cell cytosol. Finally, a COPI binding domain swap was used to demonstrate that substitution of the lysine-rich transmembrane helix 1with the COPI binding portion of the p23 adaptor cytoplasmic tail results in a mutant that displays full wild type activity. Thus, irrespective of sequence, the ability of transmembrane helix 1 to bind to COPI complex appears to be the essential feature for catalytic domain delivery to the cytosol. PMID:20398220

  1. The first seven amino acids encoded by the v-src oncogene act as a myristylation signal: Lysine 7 is a critical determinant

    SciTech Connect

    Kaplan, J.M.; Mardon, G.; Bishop, J.M.; Varmus, H.E.

    1988-06-01

    The transforming protein of Rous sarcoma virus, pp60/sup v-src/, is covalently coupled to myristic acid by an amide linkage to glycine 2. Myristylation promotes the association of pp60/sup v-src/ with cellular membranes, and this subcellular location is essential for transforming activity. The findings presented here, in conjunction with the previous reports of others, imply that the seventh amino acid encoded by v-src might be important in the myristlyation reaction. Replacement of lysine 7 by asparagine greatly reduced the myristylation, membrane association, and transforming activity of pp60/sup v-src/. In contrast, substitution of arginine at residue 7 had no effect on any of these properties of pp60/sup v-src/. Addition of amino acids 1 to 7 encoded by v-src was sufficient to cause myristylation of a src-pyruvate kinase function protein. The authors conclude that the recognition sequence for myristylation of pp60/sup v-src/ comprises amino acids 1 to 7 and that lysine 7 is a critical component of this sequence.

  2. Role of portal region lysine residues in electrostatic interactions between heart fatty acid binding protein and phospholipid membranes.

    PubMed

    Herr, F M; Aronson, J; Storch, J

    1996-01-30

    The structure of heart fatty acid binding protein (HFABP) is a flattened beta-barrel comprising 10 antiparallel beta-sheets capped by two alpha-helical segments. The helical cap region is hypothesized to behave as a portal "lid" for the entry and release of ligand from the binding pocket. The transfer of fatty acid from HFABP is thought to occur via effective collisional interactions with membranes, and these interactions are enhanced when transfer is to membranes of net negative charge, thus implying that specific basic residues on the surface of HFABP may govern the transfer process [Wootan, M. G., & Storch, J. (1994) J. Biol. Chem. 269, 10517-10523]. To directly examine the role of charged lysine residues on the HFABP surface in specific interactions with membranes, chemical modification and selective mutagenesis of HFABP were used. All surface lysine residues were neutralized by acetylation of recombinant HFABP with acetic anhydride. In addition, seven mutant HFABPs were generated that resulted in charge alterations in five distinct sites of HFABP. Modification of the protein did not significantly alter the structural or ligand binding properties of HFABP, as assessed by circular dichroism, fluorescence quantum yield, and ligand binding analyses. By using a resonance energy transfer assay, transfer of 2-(9-anthroyloxy)palmitate (2AP) from acetylated HFABP to membranes was significantly slower than transfer from native HFABP. In addition, in distinct contrast to transfer from native protein, the 2AP transfer rate from acetylated HFABP was not increased to acceptor membranes of increased negative charge. Transfer of 2AP from HFABP mutants involving K22, located on alpha-helix I (alpha-I) of the helical cap region, was 3-fold slower than transfer from wild-type protein, whereas rates from a mutant involving the K59 residue, located on the beta 2-turn of the barrel near the helical cap, were 2-fold faster than those of wild type. A double mutant involving K22 and K

  3. Identification of gunshot residue: a critical review.

    PubMed

    Saverio Romolo, F; Margot, P

    2001-06-15

    A review of the scientific papers published on inorganic gunshot residue (GSR) analysis permits to study how the particle analysis has shown its capability in detection and identification of gunshot residue. The scanning electron microscope can be the most powerful tool for forensic scientists to determine the proximity to a discharging firearm and/or the contact with a surface exposed to GSR. Particle analysis can identify individual gunshot residue particles through both morphological and elemental characteristics. When particles are detected on the collected sample, the analytical results can be interpreted following rules of a formal general interpretative system, to determine whether they come from the explosion of a primer or from other possible sources. The particles on the sample are compared with an abstract idea of "unique" GSR particle produced by the sole source of the explosion of a primer. "Uniqueness" is not the only problem related to GSR detection and identification for a forensic scientist. With "not-unique" particles interpretation of results is extremely important. The evidential strength of "not-unique" particles can increase with a more fruitful interpretative framework based on Bayes rule. For the assessment of the value of a GSR in linking a suspect and a crime, it is important to compare two hypothesis: the first can be that of the evidence if the suspect has been shooting in a specific situation, the second that of the evidence if the suspect was not involved in this shooting. This case specific or case-by-case approach is closer to what the court is interested in. The authors consider that a "case-by-case" approach should be followed whenever possible. Research of models and data such as those developed in other trace evidence material (fibres, glass, etc.) using a Bayesian approach is suggested in the interpretation of GSR. PMID:11376984

  4. A Key Role for Lysine Residues in Amyloid β-Protein Folding, Assembly, and Toxicity

    PubMed Central

    2012-01-01

    A combination of hydrophobic and electrostatic interactions is important in initiating the aberrant self-assembly process that leads to formation of toxic oligomers and aggregates by multiple disease-related proteins, including amyloid β-protein (Aβ), whose self-assembly is believed to initiate brain pathogenesis in Alzheimer’s disease. Lys residues play key roles in this process and participate in both types of interaction. They also are the target of our recently reported molecular tweezer inhibitors. To obtain further insight into the role of the two Lys residues in Aβ assembly and toxicity, here we substituted each by Ala in both Aβ40 and Aβ42 and studied the impact of the substitution on Aβ oligomerization, aggregation, and toxicity. Our data show that each substitution has a major impact on Aβ assembly and toxicity, with significant differences depending on peptide length (40 versus 42 amino acids) and the position of the substitution. In particular, Lys16→Ala substitution dramatically reduces Aβ toxicity. The data support the use of compounds targeting Lys residues specifically as inhibitors of Aβ toxicity and suggest that exploring the role of Lys residues in other disease-related amyloidogenic proteins may help understanding the mechanisms of aggregation and toxicity of these proteins. PMID:22860216

  5. High Affinity Binding of the Receptor-associated Protein D1D2 Domains with the Low Density Lipoprotein Receptor-related Protein (LRP1) Involves Bivalent Complex Formation: CRITICAL ROLES OF LYSINES 60 AND 191.

    PubMed

    Prasad, Joni M; Young, Patricia A; Strickland, Dudley K

    2016-08-26

    The LDL receptor-related protein 1 (LRP1) is a large endocytic receptor that binds and mediates the endocytosis of numerous structurally diverse ligands. Currently, the basis for ligand recognition by LRP1 is not well understood. LRP1 requires a molecular chaperone, termed the receptor-associated protein (RAP), to escort the newly synthesized receptor from the endoplasmic reticulum to the Golgi. RAP is a three-domain protein that contains the following two high affinity binding sites for LRP1: one is located within domains 1 and 2, and one is located in its third domain. Studies on the interaction of the RAP third domain with LRP1 reveal critical contributions by lysine 256 and lysine 270 for this interaction. From these studies, a model for ligand recognition by this class of receptors has been proposed. Here, we employed surface plasmon resonance to investigate the binding of RAP D1D2 to LRP1. Our results reveal that the high affinity of D1D2 for LRP1 results from avidity effects mediated by the simultaneous interactions of lysine 60 in D1 and lysine 191 in D2 with sites on LRP1 to form a bivalent D1D2-LRP1 complex. When lysine 60 and 191 are both mutated to alanine, the binding of D1D2 to LRP1 is ablated. Our data also reveal that D1D2 is able to bind to a second distinct site on LRP1 to form a monovalent complex. The studies confirm the canonical model for ligand recognition by this class of receptors, which is initiated by pairs of lysine residues that dock into acidic pockets on the receptor. PMID:27402839

  6. Dichotomy in the Epigenetic Mark Lysine Acetylation is Critical for the Proliferation of Prostate Cancer Cells

    PubMed Central

    Pathak, Ravi; Philizaire, Marc; Mujtaba, Shiraz

    2015-01-01

    The dynamics of lysine acetylation serve as a major epigenetic mark, which regulates cellular response to inflammation, DNA damage and hormonal changes. Microarray assays reveal changes in gene expression, but cannot predict regulation of a protein function by epigenetic modifications. The present study employs computational tools to inclusively analyze microarray data to understand the potential role of acetylation during development of androgen-independent PCa. The data revealed that the androgen receptor interacts with 333 proteins, out of which at least 92 proteins were acetylated. Notably, the number of cellular proteins undergoing acetylation in the androgen-dependent PCa was more as compared to the androgen-independent PCa. Specifically, the 32 lysine-acetylated proteins in the cellular models of androgen-dependent PCa were mainly involved in regulating stability as well as pre- and post-processing of mRNA. Collectively, the data demonstrate that protein lysine acetylation plays a crucial role during the transition of androgen-dependent to -independent PCa, which importantly, could also serve as a functional axis to unravel new therapeutic targets. PMID:26295410

  7. Role of an invariant lysine residue in folate binding on Escherichia coli thymidylate synthase: calorimetric and crystallographic analysis of the K48Q mutant

    PubMed Central

    Arvizu-Flores, Aldo A.; Sugich-Miranda, Rocio; Arreola, Rodrigo; Garcia-Orozco, Karina D.; Velazquez-Contreras, Enrique F.; Montfort, William R.; Maley, Frank; Sotelo-Mundo, Rogerio R.

    2008-01-01

    Thymidylate synthase (TS) catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) using methylene tetrahydrofolate (CH2THF) as cofactor, the glutamate tail of which forms a water-mediated hydrogen-bond with an invariant lysine residue of this enzyme. To understand the role of this interaction, we studied the K48Q mutant of Escherichia coli TS using structural and biophysical methods. The kcat of the K48Q mutant was 430 fold lower than wild-type TS in activity, while the the Km for the (R)-stereoisomer of CH2THF was 300 µM, about 30 fold larger than Km from the wild-type TS. Affinity constants were determined using isothermal titration calorimetry, which showed that binding was reduced by one order of magnitude for folate-like TS inhibitors, such as propargyl-dideaza folate (PDDF) or compounds that distort the TS active site like BW1843U89 (U89). The crystal structure of the K48Q-dUMP complex revealed that dUMP binding is not impaired in the mutamt, and that U89 in a ternary complex of K48Q-nucleotide-U89 was bound in the active site with subtle differences relative to comparable wild type complexes. PDDF failed to form ternary complexes with K48Q and dUMP. Thermodynamic data correlated with the structural determinations, since PDDF binding was dominated by enthalpic effects while U89 had an important entropic component. In conclusion, K48 is critical for catalysis since it leads to a productive CH2THF binding, while mutation at this residue does not affect much the binding of inhibitors that do not make contact with this group. PMID:18403248

  8. Maleimide-functionalized closo-dodecaborate albumin conjugates (MID-AC): Unique ligation at cysteine and lysine residues enables efficient boron delivery to tumor for neutron capture therapy.

    PubMed

    Kikuchi, Shunsuke; Kanoh, Daisuke; Sato, Shinichi; Sakurai, Yoshinori; Suzuki, Minoru; Nakamura, Hiroyuki

    2016-09-10

    Maleimide-conjugating closo-dodecaborate sodium form 5c (MID) synthesized by the nucleophilic ring-opening reaction of closo-dodecaborate-1,4-dioxane complex 2 with tetrabutylammonium (TBA) azide was found to conjugate to free SH of cysteine and lysine residues in BSA under physiological conditions, forming highly boronated BSA that showed high and selective accumulation in tumor and significant tumor growth inhibition in colon 26 tumor-bearing mice subjected to thermal neutron irradiation. PMID:27422608

  9. Residual Stresses and Critical Initial Flaw Size Analyses of Welds

    NASA Technical Reports Server (NTRS)

    Brust, Frederick W.; Raju, Ivatury, S.; Dawocke, David S.; Cheston, Derrick

    2009-01-01

    An independent assessment was conducted to determine the critical initial flaw size (CIFS) for the flange-to-skin weld in the Ares I-X Upper Stage Simulator (USS). A series of weld analyses are performed to determine the residual stresses in a critical region of the USS. Weld residual stresses both increase constraint and mean stress thereby having an important effect on the fatigue life. The purpose of the weld analyses was to model the weld process using a variety of sequences to determine the 'best' sequence in terms of weld residual stresses and distortions. The many factors examined in this study include weld design (single-V, double-V groove), weld sequence, boundary conditions, and material properties, among others. The results of this weld analysis are included with service loads to perform a fatigue and critical initial flaw size evaluation.

  10. Molecular Basis for Lysine Specificity in the Yeast Ubiquitin-Conjugating Enzyme Cdc34 ▿

    PubMed Central

    Sadowski, Martin; Suryadinata, Randy; Lai, Xianning; Heierhorst, Jörg; Sarcevic, Boris

    2010-01-01

    Ubiquitin (Ub)-conjugating enzymes (E2s) and ubiquitin ligases (E3s) catalyze the attachment of Ub to lysine residues in substrates and Ub during monoubiquitination and polyubiquitination. Lysine selection is important for the generation of diverse substrate-Ub structures, which provides versatility to this pathway in the targeting of proteins to different fates. The mechanisms of lysine selection remain poorly understood, with previous studies suggesting that the ubiquitination site(s) is selected by the E2/E3-mediated positioning of a lysine(s) toward the E2/E3 active site. By studying the polyubiquitination of Sic1 by the E2 protein Cdc34 and the RING E3 Skp1/Cul1/F-box (SCF) protein, we now demonstrate that in addition to E2/E3-mediated positioning, proximal amino acids surrounding the lysine residues in Sic1 and Ub are critical for ubiquitination. This mechanism is linked to key residues composing the catalytic core of Cdc34 and independent of SCF. Changes to these core residues altered the lysine preference of Cdc34 and specified whether this enzyme monoubiquitinated or polyubiquitinated Sic1. These new findings indicate that compatibility between amino acids surrounding acceptor lysine residues and key amino acids in the catalytic core of ubiquitin-conjugating enzymes is an important mechanism for lysine selection during ubiquitination. PMID:20194622

  11. Lysine post-translational modifications of collagen

    PubMed Central

    Yamauchi, Mitsuo; Sricholpech, Marnisa

    2012-01-01

    Type I collagen is the most abundant structural protein in vertebrates. It is a heterotrimeric molecule composed of two α1 chains and one α2 chain, forming a long uninterrupted triple helical structure with short non-triple helical telopeptides at both the N- and C-termini. During biosynthesis, collagen acquires a number of post-translational modifications, including lysine modifications, that are critical to the structure and biological functions of this protein. Lysine modifications of collagen are highly complicated sequential processes catalysed by several groups of enzymes leading to the final step of biosynthesis, covalent intermolecular cross-linking. In the cell, specific lysine residues are hydroxylated to form hydroxylysine. Then specific hydroxylysine residues located in the helical domain of the molecule are glycosylated by the addition of galactose or glucose-galactose. Outside the cell, lysine and hydroxylysine residues in the N- and C-telopeptides can be oxidatively deaminated to produce reactive aldehydes that undergo a series of non-enzymatic condensation reactions to form covalent intra- and inter-molecular cross-links. Owing to the recent advances in molecular and cellular biology, and analytical technologies, the biological significance and molecular mechanisms of these modifications have been gradually elucidated. This chapter provides an overview on these enzymatic lysine modifications and subsequent cross-linking. PMID:22708567

  12. Lysine carboxylation: unveiling a spontaneous post-translational modification

    SciTech Connect

    Jimenez-Morales, David; Adamian, Larisa; Shi, Dashuang; Liang, Jie

    2014-01-01

    A computational method for the prediction of lysine carboxylation (KCX) in protein structures is described. The method accurately identifies misreported KCXs and predicts previously unknown KCX sites. The carboxylation of lysine residues is a post-translational modification (PTM) that plays a critical role in the catalytic mechanisms of several important enzymes. It occurs spontaneously under certain physicochemical conditions, but is difficult to detect experimentally. Its full impact is unknown. In this work, the signature microenvironment of lysine-carboxylation sites has been characterized. In addition, a computational method called Predictor of Lysine Carboxylation (PreLysCar) for the detection of lysine carboxylation in proteins with available three-dimensional structures has been developed. The likely prevalence of lysine carboxylation in the proteome was assessed through large-scale computations. The results suggest that about 1.3% of large proteins may contain a carboxylated lysine residue. This unexpected prevalence of lysine carboxylation implies an enrichment of reactions in which it may play functional roles. The results also suggest that by switching enzymes on and off under appropriate physicochemical conditions spontaneous PTMs may serve as an important and widely used efficient biological machinery for regulation.

  13. Lysine residues direct the chlorination of tyrosines in YXXK motifs of apolipoprotein A-I when hypochlorous acid oxidizes high density lipoprotein.

    PubMed

    Bergt, Constanze; Fu, Xiaoyun; Huq, Nabiha P; Kao, Jeff; Heinecke, Jay W

    2004-02-27

    Oxidized lipoproteins may play an important role in the pathogenesis of atherosclerosis. Elevated levels of 3-chlorotyrosine, a specific end product of the reaction between hypochlorous acid (HOCl) and tyrosine residues of proteins, have been detected in atherosclerotic tissue. Thus, HOCl generated by the phagocyte enzyme myeloperoxidase represents one pathway for protein oxidation in humans. One important target of the myeloperoxidase pathway may be high density lipoprotein (HDL), which mobilizes cholesterol from artery wall cells. To determine whether activated phagocytes preferentially chlorinate specific sites in HDL, we used tandem mass spectrometry (MS/MS) to analyze apolipoprotein A-I that had been oxidized by HOCl. The major site of chlorination was a single tyrosine residue located in one of the protein's YXXK motifs (where X represents a nonreactive amino acid). To investigate the mechanism of chlorination, we exposed synthetic peptides to HOCl. The peptides encompassed the amino acid sequences YKXXY, YXXKY, or YXXXY. MS/MS analysis demonstrated that chlorination of tyrosine in the peptides that contained lysine was regioselective and occurred in high yield if the substrate was KXXY or YXXK. NMR and MS analyses revealed that the N(epsilon) amino group of lysine was initially chlorinated, which suggests that chloramine formation is the first step in tyrosine chlorination. Molecular modeling of the YXXK motif in apolipoprotein A-I demonstrated that these tyrosine and lysine residues are adjacent on the same face of an amphipathic alpha-helix. Our observations suggest that HOCl selectively targets tyrosine residues that are suitably juxtaposed to primary amino groups in proteins. This mechanism might enable phagocytes to efficiently damage proteins when they destroy microbial proteins during infection or damage host tissue during inflammation. PMID:14660678

  14. Site-Specific Labeling of Protein Lysine Residues and N-Terminal Amino Groups with Indoles and Indole-Derivatives.

    PubMed

    Larda, Sacha Thierry; Pichugin, Dmitry; Prosser, Robert Scott

    2015-12-16

    Indoles and indole-derivatives can be used to site-specifically label proteins on lysine and N-terminal amino groups under mild, nondenaturing reaction conditions. Hen egg white lysozyme (HEWL) and α-lactalbumin were labeled with indole, fluoroindole, or fluoroindole-2-carboxylate via electrophilic aromatic substitutions to lysine side chain Nε- and N-terminal amino imines, formed in situ in the presence of formaldehyde. The reaction is highly site-selective, easily controlled by temperature, and does not eliminate the native charge of the protein, unlike many other common lysine-specific labeling strategies. (19)F NMR was used to monitor reaction progression, and in the case of HEWL, unique resonances for each labeled side chain could be resolved. We demonstrate that the indole tags are highly selective for primary amino groups. (19)F NMR demonstrates that each lysine exhibits a different rate of conjugation to indoles making it possible to employ these tags as a means of probing surface topology by NMR or mass spectrometry. Given the site-specificity of this tagging method, the mildness of the reaction conditions (aqueous, buffered, or unbuffered) and the low stoichiometry required for the reaction, indole-derivatives should serve as a valuable addition to the bioconjugation toolkit. We propose that labeling lysine side chains and N-terminal amino groups with indoles is a versatile and general strategy for bioconjugations with substituted indoles having broad implications for protein functionalization. PMID:26587689

  15. The Ribosomal L1 Protuberance in Yeast Is Methylated on a Lysine Residue Catalyzed by a Seven-β-strand Methyltransferase*

    PubMed Central

    Webb, Kristofor J.; Al-Hadid, Qais; Zurita-Lopez, Cecilia I.; Young, Brian D.; Lipson, Rebecca S.; Clarke, Steven G.

    2011-01-01

    Modification of proteins of the translational apparatus is common in many organisms. In the yeast Saccharomyces cerevisiae, we provide evidence for the methylation of Rpl1ab, a well conserved protein forming the ribosomal L1 protuberance of the large subunit that functions in the release of tRNA from the exit site. We show that the intact mass of Rpl1ab is 14 Da larger than its calculated mass with the previously described loss of the initiator methionine residue and N-terminal acetylation. We determined that the increase in mass of yeast Rpl1ab is consistent with the addition of a methyl group to lysine 46 using top-down mass spectrometry. Lysine modification was confirmed by detecting 3H-N-ϵ-monomethyllysine in hydrolysates of Rpl1ab purified from yeast cells radiolabeled in vivo with S-adenosyl-l-[methyl-3H]methionine. Mass spectrometric analysis of intact Rpl1ab purified from 37 deletion strains of known and putative yeast methyltransferases revealed that only the deletion of the YLR137W gene, encoding a seven-β-strand methyltransferase, results in the loss of the +14-Da modification. We expressed the YLR137W gene as a His-tagged protein in Escherichia coli and showed that it catalyzes N-ϵ-monomethyllysine formation within Rpl1ab on ribosomes from the ΔYLR137W mutant strain lacking the methyltransferase activity but not from wild-type ribosomes. We also showed that the His-tagged protein could catalyze monomethyllysine formation on a 16-residue peptide corresponding to residues 38–53 of Rpl1ab. We propose that the YLR137W gene be given the standard name RKM5 (ribosomal lysine (K) methyltransferase 5). Orthologs of RKM5 are found only in fungal species, suggesting a role unique to their survival. PMID:21460220

  16. Preferential binding of 4-hydroxynonenal to lysine residues in specific parasite proteins in plakortin-treated Plasmodium falciparum-parasitized red blood cells

    PubMed Central

    Schwarzer, Evelin; Gallo, Valentina; Valente, Elena; Ulliers, Daniela; Taglialatela-Scafati, Orazio; Arese, Paolo; Skorokhod, Oleksii A.

    2015-01-01

    The data show the frequencies by which the amino acid residues lysine, histidine and cysteine of six proteins of the malaria parasite Plasmodium falciparum are post-translationally modified by the lipoperoxydation endproduct 4-hydroxynonenal after challenging the parasitized red blood cell with plakortin. Plakortin is an antimalarial endoperoxide whose molecular anti-parasitic effect is described in Skorokhod et al. (2015) [1]. Plakortin did not elicit hemoglobin leakage from host red blood cells and did not oxidize reduced glutathione. PMID:26702418

  17. Lysine carboxylation: unveiling a spontaneous post-translational modification

    PubMed Central

    Jimenez-Morales, David; Adamian, Larisa; Shi, Dashuang; Liang, Jie

    2014-01-01

    The carboxylation of lysine residues is a post-translational modification (PTM) that plays a critical role in the catalytic mechanisms of several important enzymes. It occurs spontaneously under certain physicochemical conditions, but is difficult to detect experimentally. Its full impact is unknown. In this work, the signature microenvironment of lysine-carboxylation sites has been characterized. In addition, a computational method called Predictor of Lysine Carboxyl­ation (PreLysCar) for the detection of lysine carboxylation in proteins with available three-dimensional structures has been developed. The likely prevalence of lysine carboxylation in the proteome was assessed through large-scale computations. The results suggest that about 1.3% of large proteins may contain a carboxylated lysine residue. This unexpected prevalence of lysine carboxylation implies an enrichment of reactions in which it may play functional roles. The results also suggest that by switching enzymes on and off under appropriate physicochemical conditions spontaneous PTMs may serve as an important and widely used efficient biological machinery for regulation. PMID:24419378

  18. Roles of Arginine and Lysine Residues in the Translocation of a Cell-Penetrating Peptide from 13C, 31P and 19F Solid-State NMR

    PubMed Central

    Su, Yongchao; Doherty, Tim; Waring, Alan J.; Ruchala, Piotr; Hong, Mei

    2009-01-01

    Cell-penetrating peptides (CPPs) are small cationic peptides that cross the cell membrane while carrying macromolecular cargoes. We use solid-state NMR to investigate the structure and lipid interaction of two cationic residues, Arg10 and Lys13, in the CPP penetratin. 13C chemical shifts indicate that Arg10 adopts a rigid β-strand conformation in the liquid-crystalline state of anionic lipid membranes. This behavior contrasts with all other residues observed so far in this peptide, which adopt a dynamic β-turn conformation with coil-like chemical shifts at physiological temperature. Low-temperature 13C-31P distances between the peptide and the lipid phosphates indicate that both the Arg10 guanidinium Cζ and the Lys13 Cε lie in close proximity to the lipid 31P (4.0 - 4.2 Å), proving the existence of charge-charge interaction for both Arg10 and Lys13 in the gel-phase membrane. However, since lysine substitution in CPPs are known to reduce their translocation ability, we propose that low temperature stabilizes both lysine and arginine interactions with the phosphates, whereas at high temperature the lysine-phosphate interaction is much weaker than the arginine-phosphate interaction. This is supported by the unusually high rigidity of the Arg10 sidechain and its β-strand conformation at high temperature. The latter is proposed to be important for ion pair formation by allowing close approach of the lipid headgroups to guanidinium sidechains. 19F and 13C spin diffusion experiments indicate that penetratin is oligomerized into β-sheets in gel-phase membranes. These solid-state NMR data indicate that guanidinium-phosphate interactions exist in penetratin, and guanidinium groups play a stronger structural role than ammonium groups in the lipid-assisted translocation of CPPs across liquid-crystalline cell membranes. PMID:19364134

  19. The ɛ-Amino Group of Protein Lysine Residues Is Highly Susceptible to Nonenzymatic Acylation by Several Physiological Acyl-CoA Thioesters.

    PubMed

    Simic, Zeljko; Weiwad, Matthias; Schierhorn, Angelika; Steegborn, Clemens; Schutkowski, Mike

    2015-11-01

    Mitochondrial enzymes implicated in the pathophysiology of diabetes, cancer, and metabolic syndrome are highly regulated by acetylation. However, mitochondrial acetyltransferases have not been identified. Here, we show that acetylation and also other acylations are spontaneous processes that depend on pH value, acyl-CoA concentration and the chemical nature of the acyl residue. In the case of a peptide derived from carbamoyl phosphate synthetase 1, the rates of succinylation and glutarylation were up to 150 times than for acetylation. These results were confirmed by using the protein substrate cyclophilin A (CypA). Deacylation experiments revealed that SIRT3 exhibits deacetylase activity but is not able to remove any of the succinyl groups from CypA, whereas SIRT5 is an effective protein desuccinylase. Thus, the acylation landscape on lysine residues might largely depend on the enzymatic activity of specific sirtuins, and the availability and reactivity of acyl-CoA compounds. PMID:26382620

  20. A Jump-from-Cavity Pyrophosphate Ion Release Assisted by a Key Lysine Residue in T7 RNA Polymerase Transcription Elongation

    PubMed Central

    Da, Lin-Tai; E, Chao; Duan, Baogen; Zhang, Chuanbiao; Zhou, Xin; Yu, Jin

    2015-01-01

    Pyrophosphate ion (PPi) release during transcription elongation is a signature step in each nucleotide addition cycle. The kinetics and energetics of the process as well as how it proceeds with substantial conformational changes of the polymerase complex determine the mechano-chemical coupling mechanism of the transcription elongation. Here we investigated detailed dynamics of the PPi release process in a single-subunit RNA polymerase (RNAP) from bacteriophage T7, implementing all-atom molecular dynamics (MD) simulations. We obtained a jump-from-cavity kinetic model of the PPi release utilizing extensive nanosecond MD simulations. We found that the PPi release in T7 RNAP is initiated by the PPi dissociation from two catalytic aspartic acids, followed by a comparatively slow jump-from-cavity activation process. Combining with a number of microsecond long MD simulations, we also found that the activation process is hindered by charged residue associations as well as by local steric and hydrogen bond interactions. On the other hand, the activation is greatly assisted by a highly flexible lysine residue Lys472 that swings its side chain to pull PPi out. The mechanism can apply in general to single subunit RNA and DNA polymerases with similar molecular structures and conserved key residues. Remarkably, the flexible lysine or arginine residue appears to be a universal module that assists the PPi release even in multi-subunit RNAPs with charge facilitated hopping mechanisms. We also noticed that the PPi release is not tightly coupled to opening motions of an O-helix on the fingers domain of T7 RNAP according to the microsecond MD simulations. Our study thus supports the Brownian ratchet scenario of the mechano-chemical coupling in the transcription elongation of the single-subunit polymerase. PMID:26599007

  1. A Jump-from-Cavity Pyrophosphate Ion Release Assisted by a Key Lysine Residue in T7 RNA Polymerase Transcription Elongation.

    PubMed

    Da, Lin-Tai; E, Chao; Duan, Baogen; Zhang, Chuanbiao; Zhou, Xin; Yu, Jin

    2015-11-01

    Pyrophosphate ion (PPi) release during transcription elongation is a signature step in each nucleotide addition cycle. The kinetics and energetics of the process as well as how it proceeds with substantial conformational changes of the polymerase complex determine the mechano-chemical coupling mechanism of the transcription elongation. Here we investigated detailed dynamics of the PPi release process in a single-subunit RNA polymerase (RNAP) from bacteriophage T7, implementing all-atom molecular dynamics (MD) simulations. We obtained a jump-from-cavity kinetic model of the PPi release utilizing extensive nanosecond MD simulations. We found that the PPi release in T7 RNAP is initiated by the PPi dissociation from two catalytic aspartic acids, followed by a comparatively slow jump-from-cavity activation process. Combining with a number of microsecond long MD simulations, we also found that the activation process is hindered by charged residue associations as well as by local steric and hydrogen bond interactions. On the other hand, the activation is greatly assisted by a highly flexible lysine residue Lys472 that swings its side chain to pull PPi out. The mechanism can apply in general to single subunit RNA and DNA polymerases with similar molecular structures and conserved key residues. Remarkably, the flexible lysine or arginine residue appears to be a universal module that assists the PPi release even in multi-subunit RNAPs with charge facilitated hopping mechanisms. We also noticed that the PPi release is not tightly coupled to opening motions of an O-helix on the fingers domain of T7 RNAP according to the microsecond MD simulations. Our study thus supports the Brownian ratchet scenario of the mechano-chemical coupling in the transcription elongation of the single-subunit polymerase. PMID:26599007

  2. Coactosin-like protein, a human F-actin-binding protein: critical role of lysine-75.

    PubMed Central

    Provost, P; Doucet, J; Stock, A; Gerisch, G; Samuelsson, B; Rådmark, O

    2001-01-01

    Coactosin-like protein (CLP) was recently identified in a yeast two-hybrid screen using 5-lipoxygenase as bait. In the present study, we report the functional characterization of CLP as a human filamentous actin (F-actin)-binding protein. CLP mRNA shows a wide tissue distribution and is predominantly expressed in placenta, lung, kidney and peripheral-blood leucocytes. Endogenous CLP is localized in the cytosol of myeloid cells. Using a two-hybrid approach, actin was identified as a CLP-interacting protein. Binding experiments indicated that CLP associates with F-actin, but does not form a stable complex with globular actin. In transfected mammalian cells, CLP co-localized with actin stress fibres. CLP bound to actin filaments with a stoichiometry of 1:2 (CLP: actin subunits), but could be cross-linked to only one subunit of actin. Site-directed mutagenesis revealed the involvement of Lys(75) of CLP in actin binding, a residue highly conserved in related proteins and supposed to be exposed on the surface of the CLP protein. Our results identify CLP as a new human protein that binds F-actin in vitro and in vivo, and indicate that Lys(75) is essential for this interaction. PMID:11583571

  3. Endoplasmic reticulum-associated degradation of Niemann-Pick C1: evidence for the role of heat shock proteins and identification of lysine residues that accept ubiquitin.

    PubMed

    Nakasone, Naoe; Nakamura, Yuko S; Higaki, Katsumi; Oumi, Nao; Ohno, Kousaku; Ninomiya, Haruaki

    2014-07-11

    Most cases with Niemann-Pick disease type C carry mutations in NPC1. Some of the mutations, including the most frequent I1061T, give rise to unstable proteins selected for endoplasmic reticulum-associated degradation. The purpose of the current study was to shed mechanistic insights into the degradation process. A proteasome inhibitor MG132 prolonged the life span of the wild-type NPC1 expressed in COS cells. The expressed protein associated with multiple chaperones including heat shock protein 90 (Hsp90), Hsp70, heat shock cognate protein 70 (Hsc70), and calnexin. Accordingly, expression of an E3 ligase CHIP (carboxyl terminus of Hsp70-interacting protein) enhanced MG132-induced accumulation of ubiquitylated NPC1. Co-expression and RNAi knockdown experiments in HEK cells indicated that Hsp70/Hsp90 stabilized NPC1, whereas Hsc70 destabilized it. In human fibroblasts carrying the I1061T mutation, adenovirus-mediated expression of Hsp70 or treatment with an HSP-inducer geranylgeranylacetone (GGA) increased the level of the mutant protein. In GGA-treated cells, the rescued protein was localized in the late endosome and ameliorated cholesterol accumulation. MALDI-TOF mass spectrometry revealed three lysine residues at amino acids 318, 792, and 1180 as potential ubiquitin-conjugation sites. Substitutions of the three residues with alanine yielded a mutant protein with a steady-state level more than three times higher than that of the wild-type. Introduction of the same substitutions to the I1061T mutant resulted in an increase in its protein level and functional restoration. These findings indicated the role of HSPs in quality control of NPC1 and revealed the role of three lysine residues as ubiquitin-conjugation sites. PMID:24891511

  4. Lysine221 is the general base residue of the isochorismate synthase from Pseudomonas aeruginosa (PchA) in a reaction that is diffusion limited.

    PubMed

    Meneely, Kathleen M; Luo, Qianyi; Dhar, Prajnaparamita; Lamb, Audrey L

    2013-10-01

    The isochorismate synthase from Pseudomonas aeruginosa (PchA) catalyzes the conversion of chorismate to isochorismate, which is subsequently converted by a second enzyme (PchB) to salicylate for incorporation into the salicylate-capped siderophore pyochelin. PchA is a member of the MST family of enzymes, which includes the structurally homologous isochorismate synthases from Escherichia coli (EntC and MenF) and salicylate synthases from Yersinia enterocolitica (Irp9) and Mycobacterium tuberculosis (MbtI). The latter enzymes generate isochorismate as an intermediate before generating salicylate and pyruvate. General acid-general base catalysis has been proposed for isochorismate synthesis in all five enzymes, but the residues required for the isomerization are a matter of debate, with both lysine221 and glutamate313 proposed as the general base (PchA numbering). This work includes a classical characterization of PchA with steady state kinetic analysis, solvent kinetic isotope effect analysis and by measuring the effect of viscosogens on catalysis. The results suggest that isochorismate production from chorismate by the MST enzymes is the result of general acid-general base catalysis with a lysine as the base and a glutamic acid as the acid, in reverse protonation states. Chemistry is determined to not be rate limiting, favoring the hypothesis of a conformational or binding step as the slow step. PMID:23942051

  5. Conserved residue lysine165 is essential for the ability of O6-alkylguanine-DNA alkyltransferase to react with O6-benzylguanine.

    PubMed Central

    Xu-Welliver, M; Kanugula, S; Loktionova, N A; Crone, T M; Pegg, A E

    2000-01-01

    The role of lysine(165) in the activity of the DNA repair protein, O(6)-alkylguanine-DNA alkyltransferase (AGT), and the ability of AGT to react with the pseudosubstrate inhibitor, O(6)-benzylguanine (BG), was investigated by changing this lysine to all other 19 possibilities. All of these mutants (except for K165T, which could not be tested as it was too poorly active for assay in crude cell extracts) gave BG-resistant AGTs with increases in the amount of inhibitor needed to produce a 50% loss of activity in a 30 min incubation (ED(50)) from 100-fold (K165A) to 2400-fold (K165F). Lys(165) is a completely conserved residue in AGTs from many species, and all of the mutations at this site also reduced the ability to repair methylated DNA. The least deleterious change was that to arginine, which reduced the rate constant for DNA repair by approx. 2.5-fold. Mutant K165R resembled all of the other mutants in being highly resistant to BG, with an ED(50) value for inactivation by BG>200-fold greater than wild-type. Detailed studies of purified K165A AGT showed that the rate constant for repair and the binding to methylated DNA substrates were reduced by 10-20-fold. Despite this, the K165A mutant AGT was able to protect cells from alkylating agents and this protection was not abolished by BG. These results show that, firstly, lysine at position 165 is needed for optimal activity of AGT towards methylated DNA substrates and is essential for efficient reaction with BG; and second, even if the AGT activity towards methylated DNA substrates is impaired by mutations at codon 165, such mutants can protect tumour cells from therapeutic alkylating agents. These results raise the possibility that the conservation of Lys(165) is due to the need for AGT activity towards substrates containing more bulky adducts than O(6)-methylguanine. They also suggest that alterations at Lys(165) may occur during chemotherapy with BG and alkylating agents and could limit the effectiveness of this

  6. Acetylation and glycation of fibrinogen in vitro occur at specific lysine residues in a concentration dependent manner: A mass spectrometric and isotope labeling study

    SciTech Connect

    Svensson, Jan; Bergman, Ann-Charlotte; Adamson, Ulf; Blombaeck, Margareta; Wallen, Hakan; Joerneskog, Gun

    2012-05-04

    Highlights: Black-Right-Pointing-Pointer Fibrinogen was incubated in vitro with glucose or aspirin. Black-Right-Pointing-Pointer Acetylations and glycations were found at twelve lysine sites by mass spectrometry. Black-Right-Pointing-Pointer The labeling by aspirin and glucose occurred dose-dependently. Black-Right-Pointing-Pointer No competition between glucose and aspirin for binding to fibrinogen was found. -- Abstract: Aspirin may exert part of its antithrombotic effects through platelet-independent mechanisms. Diabetes is a condition in which the beneficial effects of aspirin are less prominent or absent - a phenomenon called 'aspirin resistance'. We investigated whether acetylation and glycation occur at specific sites in fibrinogen and if competition between glucose and aspirin in binding to fibrinogen occurs. Our hypothesis was that such competition might be one explanation to 'aspirin resistance' in diabetes. After incubation of fibrinogen in vitro with aspirin (0.8 mM, 24 h) or glucose (100 mM, 5-10 days), we found 12 modified sites with mass spectrometric techniques. Acetylations in the {alpha}-chain: {alpha}K191, {alpha}K208, {alpha}K224, {alpha}K429, {alpha}K457, {alpha}K539, {alpha}K562, in the {beta}-chain: {beta}K233, and in the {gamma}-chain: {gamma}K170 and {gamma}K273. Glycations were found at {beta}K133 and {gamma}K75, alternatively {gamma}K85. Notably, the lysine 539 is a site involved in FXIII-mediated cross-linking of fibrin. With isotope labeling in vitro, using [{sup 14}C-acetyl]salicylic acid and [{sup 14}C]glucose, a labeling of 0.013-0.084 and 0.12-0.5 mol of acetylated and glycated adduct/mol fibrinogen, respectively, was found for clinically (12.9-100 {mu}M aspirin) and physiologically (2-8 mM glucose) relevant plasma concentrations. No competition between acetylation and glycation could be demonstrated. Thus, fibrinogen is acetylated at several lysine residues, some of which are involved in the cross-linking of fibrinogen. This may

  7. Characterization of oxidation products from 1-palmitoyl-2-linoleoyl-sn-glycerophosphatidylcholine in aqueous solutions and their reactions with cysteine, histidine and lysine residues.

    PubMed

    Milic, Ivana; Fedorova, Maria; Teuber, Kristin; Schiller, Jürgen; Hoffmann, Ralf

    2012-02-01

    This report focuses on studies of lipid peroxidation products reactivity towards the side chains of cysteine, histidine, and lysine residues in structurally unordered peptides. Thus we have analyzed linoleic acid peroxidation products (LaPP) obtained by incubating 1-palmitoyl-2-linoleoyl-sn-glycerophosphatidylcholine (PLPC) overnight with or without H(2)O(2) in the presence or absence of CuCl. In total, 55 different LaPP were identified with 26 containing reactive carbonyl groups. The strongest oxidation conditions (H(2)O(2) and Cu(I), i.e. a Fenton-like reagent) yielded 51 LaPP, whereas air oxidation produced only 12 LaPP. Independent of the oxidation conditions, around half of all LaPP were short-chain (oxidative cleavage) and the others long-chain (oxygen addition) PLPC oxidation products. The stronger oxidation conditions increased the number of LaPP, but also oxidized the added peptide Ac-PAAPAAPAPAEXTPV-OH (X=Cys, His or Lys) very quickly, especially under Fenton conditions. Thus, PLPC was oxidized by milder conditions (air or Cu(I)), incubated with the peptide and the peptide modifications were then analyzed by nano-RPC-ESI-Orbitrap-MS. Ten LaPP-derived peptide modifications were identified at lysine, whereas nine products were identified for cysteine and only three for histidine. Three high molecular weight LaPP still esterified to the GPC backbone were detected on Lys-containing peptide. Furthermore, three LaPP-derived mass shifts were obtained at cysteine, which have not previously been reported. PMID:22222463

  8. SIRT1 Regulates Thyroid-Stimulating Hormone Release by Enhancing PIP5Kγ Activity through Deacetylation of Specific Lysine Residues in Mammals

    PubMed Central

    Akieda-Asai, Sayaka; Zaima, Nobuhiro; Ikegami, Koji; Kahyo, Tomoaki; Yao, Ikuko; Hatanaka, Takahiro; Iemura, Shun-ichiro; Sugiyama, Rika; Yokozeki, Takeaki; Eishi, Yoshinobu; Koike, Morio; Ikeda, Kyoji; Chiba, Takuya; Yamaza, Haruyoshi; Shimokawa, Isao; Song, Si-Young; Matsuno, Akira; Mizutani, Akiko; Sawabe, Motoji; Chao, Moses V.; Tanaka, Masashi; Kanaho, Yasunori; Natsume, Tohru; Sugimura, Haruhiko; Date, Yukari; McBurney, Michael W.; Guarente, Leonard; Setou, Mitsutoshi

    2010-01-01

    Background SIRT1, a NAD-dependent deacetylase, has diverse roles in a variety of organs such as regulation of endocrine function and metabolism. However, it remains to be addressed how it regulates hormone release there. Methodology/Principal Findings Here, we report that SIRT1 is abundantly expressed in pituitary thyrotropes and regulates thyroid hormone secretion. Manipulation of SIRT1 level revealed that SIRT1 positively regulated the exocytosis of TSH-containing granules. Using LC/MS-based interactomics, phosphatidylinositol-4-phosphate 5-kinase (PIP5K)γ was identified as a SIRT1 binding partner and deacetylation substrate. SIRT1 deacetylated two specific lysine residues (K265/K268) in PIP5Kγ and enhanced PIP5Kγ enzyme activity. SIRT1-mediated TSH secretion was abolished by PIP5Kγ knockdown. SIRT1 knockdown decreased the levels of deacetylated PIP5Kγ, PI(4,5)P2, and reduced the secretion of TSH from pituitary cells. These results were also observed in SIRT1-knockout mice. Conclusions/Significance Our findings indicated that the control of TSH release by the SIRT1-PIP5Kγ pathway is important for regulating the metabolism of the whole body. PMID:20668706

  9. Nondestructive Testing Residual Stress Using Ultrasonic Critical Refracted Longitudinal Wave

    NASA Astrophysics Data System (ADS)

    Xu, Chunguang; Song, Wentao; Pan, Qinxue; Li, Huanxin; Liu, Shuai

    Residual stress has significant impacts on the performance of the mechanical components, especially on its strength, fatigue life and corrosion resistance and dimensional stability. Based on theory of acoustoelasticity, the testing principle of ultrasonic LCR wave method is analyzed. The testing system of residual stress is build. The method of calibration of stress coefficient is proposed in order to improve the detection precision. At last, through experiments and applications on residual stress testing of oil pipeline weld joint, vehicle's torsion shaft, glass and ceramics, gear tooth root, and so on, the result show that it deserved to be studied deeply on application and popularization of ultrasonic LCR wave method.

  10. Fragmentation behavior of Amadori-peptides obtained by non-enzymatic glycosylation of lysine residues with ADP-ribose in tandem mass spectrometry.

    PubMed

    Fedorova, Maria; Frolov, Andrej; Hoffmann, Ralf

    2010-06-01

    Mono- and poly-adenosine diphosphate (ADP)-ribosylation are common post-translational modifications incorporated by sequence-specific enzymes at, predominantly, arginine, asparagine, glutamic acid or aspartic acid residues, whereas non-enzymatic ADP-ribosylation (glycation) modifies lysine and cysteine residues. These glycated proteins and peptides (Amadori-compounds) are commonly found in organisms, but have so far not been investigated to any great degree. In this study, we have analyzed their fragmentation characteristics using different mass spectrometry (MS) techniques. In matrix-assisted laser desorption/ionization (MALDI)-MS, the ADP-ribosyl group was cleaved, almost completely, at the pyrophosphate bond by in-source decay. In contrast, this cleavage was very weak in electrospray ionization (ESI)-MS. The same fragmentation site also dominated the MALDI-PSD (post-source decay) and ESI-CID (collision-induced dissociation) mass spectra. The remaining phospho-ribosyl group (formed by the loss of adenosine monophosphate) was stable, providing a direct and reliable identification of the modification site via the b- and y-ion series. Cleavage of the ADP-ribose pyrophosphate bond under CID conditions gives access to both neutral loss (347.10 u) and precursor-ion scans (m/z 348.08), and thereby permits the identification of ADP-ribosylated peptides in complex mixtures with high sensitivity and specificity. With electron transfer dissociation (ETD), the ADP-ribosyl group was stable, providing ADP-ribosylated c- and z-ions, and thus allowing reliable sequence analyses. PMID:20527035

  11. Nucleolar accumulation of APE1 depends on charged lysine residues that undergo acetylation upon genotoxic stress and modulate its BER activity in cells

    PubMed Central

    Lirussi, Lisa; Antoniali, Giulia; Vascotto, Carlo; D'Ambrosio, Chiara; Poletto, Mattia; Romanello, Milena; Marasco, Daniela; Leone, Marilisa; Quadrifoglio, Franco; Bhakat, Kishor K.; Scaloni, Andrea; Tell, Gianluca

    2012-01-01

    Apurinic/apyrimidinic endonuclease 1 (APE1) is the main abasic endonuclease in the base excision repair (BER) pathway of DNA lesions caused by oxidation/alkylation in mammalian cells; within nucleoli it interacts with nucleophosmin and rRNA through N-terminal Lys residues, some of which (K27/K31/K32/K35) may undergo acetylation in vivo. Here we study the functional role of these modifications during genotoxic damage and their in vivo relevance. We demonstrate that cells expressing a specific K-to-A multiple mutant are APE1 nucleolar deficient and are more resistant to genotoxic treatment than those expressing the wild type, although they show impaired proliferation. Of interest, we find that genotoxic treatment induces acetylation at these K residues. We also find that the charged status of K27/K31/K32/K35 modulates acetylation at K6/K7 residues that are known to be involved in the coordination of BER activity through a mechanism regulated by the sirtuin 1 deacetylase. Of note, structural studies show that acetylation at K27/K31/K32/K35 may account for local conformational changes on APE1 protein structure. These results highlight the emerging role of acetylation of critical Lys residues in regulating APE1 functions. They also suggest the existence of cross-talk between different Lys residues of APE1 occurring upon genotoxic damage, which may modulate APE1 subnuclear distribution and enzymatic activity in vivo. PMID:22918947

  12. IDENTIFYING CRITICAL CYSTEINE RESIDUES IN ARSENIC (+3 OXIDATION STATE) METHYLTRANSFERASE

    EPA Science Inventory

    Arsenic (+3 oxidation state) methyltransferase (AS3MT) catalyzes methylation of inorganic arsenic to mono, di, and trimethylated arsenicals. Orthologous AS3MT genes in genomes ranging from simple echinoderm to human predict a protein with five conserved cysteine (C) residues. In ...

  13. The valine and lysine residues in the conserved FxVTxK motif are important for the function of phylogenetically distant plant cellulose synthases.

    PubMed

    Slabaugh, Erin; Scavuzzo-Duggan, Tess; Chaves, Arielle; Wilson, Liza; Wilson, Carmen; Davis, Jonathan K; Cosgrove, Daniel J; Anderson, Charles T; Roberts, Alison W; Haigler, Candace H

    2016-05-01

    Cellulose synthases (CESAs) synthesize the β-1,4-glucan chains that coalesce to form cellulose microfibrils in plant cell walls. In addition to a large cytosolic (catalytic) domain, CESAs have eight predicted transmembrane helices (TMHs). However, analogous to the structure of BcsA, a bacterial CESA, predicted TMH5 in CESA may instead be an interfacial helix. This would place the conserved FxVTxK motif in the plant cell cytosol where it could function as a substrate-gating loop as occurs in BcsA. To define the functional importance of the CESA region containing FxVTxK, we tested five parallel mutations in Arabidopsis thaliana CESA1 and Physcomitrella patens CESA5 in complementation assays of the relevant cesa mutants. In both organisms, the substitution of the valine or lysine residues in FxVTxK severely affected CESA function. In Arabidopsis roots, both changes were correlated with lower cellulose anisotropy, as revealed by Pontamine Fast Scarlet. Analysis of hypocotyl inner cell wall layers by atomic force microscopy showed that two altered versions of Atcesa1 could rescue cell wall phenotypes observed in the mutant background line. Overall, the data show that the FxVTxK motif is functionally important in two phylogenetically distant plant CESAs. The results show that Physcomitrella provides an efficient model for assessing the effects of engineered CESA mutations affecting primary cell wall synthesis and that diverse testing systems can lead to nuanced insights into CESA structure-function relationships. Although CESA membrane topology needs to be experimentally determined, the results support the possibility that the FxVTxK region functions similarly in CESA and BcsA. PMID:26646446

  14. Atomic structure of recombinant thaumatin II reveals flexible conformations in two residues critical for sweetness and three consecutive glycine residues.

    PubMed

    Masuda, Tetsuya; Mikami, Bunzo; Tani, Fumito

    2014-11-01

    Thaumatin, an intensely sweet-tasting protein used as a sweetener, elicits a sweet taste at 50 nM. Although two major variants designated thaumatin I and thaumatin II exist in plants, there have been few dedicated thaumatin II structural studies and, to date, data beyond atomic resolution had not been obtained. To identify the detailed structural properties explaining why thaumatin elicits a sweet taste, the structure of recombinant thaumatin II was determined at the resolution of 0.99 Å. Atomic resolution structural analysis with riding hydrogen atoms illustrated the differences in the direction of the side-chains more precisely and the electron density maps of the C-terminal regions were markedly improved. Though it had been suggested that the three consecutive glycine residues (G142-G143-G144) have highly flexible conformations, G143, the central glycine residue was successfully modelled in two conformations for the first time. Furthermore, the side chain r.m.s.d. values for two residues (R67 and R82) critical for sweetness exhibited substantially higher values, suggesting that these residues are highly disordered. These results demonstrated that the flexible conformations in two critical residues favoring their interaction with sweet taste receptors are prominent features of the intensely sweet taste of thaumatin. PMID:25066915

  15. A statistical approach to determining criticality of residual host cell DNA.

    PubMed

    Yang, Harry; Wei, Ziping; Schenerman, Mark

    2015-01-01

    We propose a method for determining the criticality of residual host cell DNA, which is characterized through two attributes, namely the size and amount of residual DNA in biopharmaceutical product. By applying a mechanistic modeling approach to the problem, we establish the linkage between residual DNA and product safety measured in terms of immunogenicity, oncogenicity, and infectivity. Such a link makes it possible to establish acceptable ranges of residual DNA size and amount. Application of the method is illustrated through two real-life examples related to a vaccine manufactured in Madin Darby Canine Kidney cell line and a monoclonal antibody using Chinese hamster ovary (CHO) cell line as host cells. PMID:25358029

  16. Tackling Critical Catalytic Residues in Helicobacter pylori l-Asparaginase

    PubMed Central

    Maggi, Maristella; Chiarelli, Laurent R; Valentini, Giovanna; Scotti, Claudia

    2015-01-01

    Bacterial asparaginases (amidohydrolases, EC 3.5.1.1) are important enzymes in cancer therapy, especially for Acute Lymphoblastic Leukemia. They are tetrameric enzymes able to catalyze the deamination of l-ASN and, to a variable extent, of l-GLN, on which leukemia cells are dependent for survival. In contrast to other known l-asparaginases, Helicobacter pylori CCUG 17874 type II enzyme (HpASNase) is cooperative and has a low affinity towards l-GLN. In this study, some critical amino acids forming the active site of HpASNase (T16, T95 and E289) have been tackled by rational engineering in the attempt to better define their role in catalysis and to achieve a deeper understanding of the peculiar cooperative behavior of this enzyme. Mutations T16E, T95D and T95H led to a complete loss of enzymatic activity. Mutation E289A dramatically reduced the catalytic activity of the enzyme, but increased its thermostability. Interestingly, E289 belongs to a loop that is very variable in l-asparaginases from the structure, sequence and length point of view, and which could be a main determinant of their different catalytic features. PMID:25826146

  17. The histone H3 lysine-27 demethylase Jmjd3 plays a critical role in specific regulation of Th17 cell differentiation.

    PubMed

    Liu, Zhi; Cao, Wei; Xu, Longxia; Chen, Xi; Zhan, Yu; Yang, Qian; Liu, Sanhong; Chen, Pengfei; Jiang, Yuhang; Sun, Xiaohua; Tao, Yu; Hu, Yiming; Li, Cuifeng; Wang, Qi; Wang, Ying; Chen, Charlie Degui; Shi, Yufang; Zhang, Xiaoren

    2015-12-01

    Interleukin (IL) 17-producing T helper (Th17) cells play critical roles in the clearance of extracellular bacteria and fungi as well as the pathogenesis of various autoimmune diseases, such as multiple sclerosis, psoriasis, and ulcerative colitis. Although a global transcriptional regulatory network of Th17 cell differentiation has been mapped recently, the participation of epigenetic modifications in the differentiation process has yet to be elucidated. We demonstrated here that histone H3 lysine-27 (H3K27) demethylation, predominantly mediated by the H3K27 demethylase Jmjd3, crucially regulated Th17 cell differentiation. Activation of naïve CD4(+) T cells immediately induced high expression of Jmjd3. Genetic depletion of Jmjd3 in CD4(+) T cells specifically impaired Th17 cell differentiation both in vitro and in vivo. Ectopic expression of Jmjd3 largely rescued the impaired differentiation of Th17 cells in vitro in Jmjd3-deficient CD4(+) T cells. Importantly, Jmjd3-deficient mice were resistant to the induction of experimental autoimmune encephalomyelitis (EAE). Furthermore, inhibition of the H3K27 demethylase activity with the specific inhibitor GSK-J4 dramatically suppressed Th17 cell differentiation in vitro. At the molecular level, Jmjd3 directly bound to and reduced the level of H3K27 trimethylation (me3) at the genomic sites of Rorc, which encodes the master Th17 transcription factor Rorγt, and Th17 cytokine genes such as Il17, Il17f, and Il22. Therefore, our studies established a critical role of Jmjd3-mediated H3K27 demethylation in Th17 cell differentiation and suggest that Jmjd3 can be a novel therapeutic target for suppressing autoimmune responses. PMID:25840993

  18. Global profiling of lysine acetylation in human histoplasmosis pathogen Histoplasma capsulatum.

    PubMed

    Xie, Longxiang; Fang, Wenjie; Deng, Wanyan; Yu, Zhaoxiao; Li, Juan; Chen, Min; Liao, Wanqing; Xie, Jianping; Pan, Weihua

    2016-04-01

    Histoplasma capsulatum is the causative agent of human histoplasmosis, which can cause respiratory and systemic mycosis in immune-compromised individuals. Lysine acetylation, a protein posttranslational protein modification, is widespread in both eukaryotes and prokaryotes. Although increasing evidence suggests that lysine acetylation may play critical roles in fungus physiology, very little is known about its extent and function in H. capsulatum. To comprehensively profile protein lysine acetylation in H. capsulatum, we performed a global acetylome analysis through peptide prefractionation, antibody enrichment, and LC-MS/MS analysis, identifying 775 acetylation sites on 456 acetylated proteins; and functionally analysis showing their involvement in different biological processes. We defined six types of acetylation site motifs, and the results imply that lysine residue of polypeptide with tyrosine at the -1 and +1 positions, histidine at the +1 position, and phenylalanine (F) at the +1 and +2 position is a preferred substrate of lysine acetyltransferase. Moreover, some virulence factors candidates including calmodulin and DnaK are acetylated. In conclusion, our data set may serve as an important resource for the elucidation of associations between functional protein lysine acetylation and virulence in H. capsulatum. PMID:26806293

  19. Critical considerations in the mitigation of insect residue contamination on aircraft surfaces - A review

    NASA Astrophysics Data System (ADS)

    Kok, Mariana; Smith, Joseph G.; Wohl, Christopher J.; Siochi, Emilie J.; Young, Trevor M.

    2015-05-01

    Mitigation of insect residue contamination on next generation aircraft is vital for the commercial exploitation of laminar flow technologies. A review of the critical entomological, meteorological and aeronautical factors affecting insect residue accumulation on aircraft leading edge surfaces is herein presented. An evaluation of a passive mitigation strategy, namely the use of anti-contamination coatings, has been conducted and the key issues in the use of these coatings highlighted. A summary of the variations in major experiments, including laboratory, wind tunnel and flight testing, is outlined. The effects of surface and material characteristics on insect residue adhesion were also investigated, with topographical features of the surface and surface chemistry shown as influential factors. The use of a substitute as an alternative to live insect testing has shown promise.

  20. Critical POU domain residues confer Oct4 uniqueness in somatic cell reprogramming.

    PubMed

    Jin, Wensong; Wang, Lei; Zhu, Fei; Tan, Weiqi; Lin, Wei; Chen, Dahua; Sun, Qinmiao; Xia, Zongping

    2016-01-01

    The POU domain transcription factor Oct4 plays critical roles in self-renewal and pluripotency of embryonic stem cells (ESCs). Together with Sox2, Klf4 and c-Myc, Oct4 can reprogram any other cell types to pluripotency, in which Oct4 is the only factor that cannot be functionally replaced by other POU family members. To investigate the determinant elements of Oct4 uniqueness, we performed Ala scan on all Ser, Thr, Tyr, Lys and Arg of murine Oct4 by testing their capability in somatic cell reprogramming. We uncovered a series of residues that are important for Oct4 functionality, in which almost all of these key residues are within the POU domains making direct interaction with DNA. The Oct4 N- and C-terminal transactivation domains (TADs) are not unique and could be replaced by the Yes-associated protein (YAP) TAD domain to support reprogramming. More importantly, we uncovered two important residues that confer Oct4 uniqueness in somatic cell reprogramming. Our systematic structure-function analyses bring novel mechanistic insight into the molecular basis of how critical residues function together to confer Oct4 uniqueness among POU family for somatic cell reprogramming. PMID:26877091

  1. Interplay between lysine methylation and Cdk phosphorylation in growth control by the retinoblastoma protein

    PubMed Central

    Carr, Simon M; Munro, Shonagh; Kessler, Benedikt; Oppermann, Udo; La Thangue, Nicholas B

    2011-01-01

    As a critical target for cyclin-dependent kinases (Cdks), the retinoblastoma tumour suppressor protein (pRb) controls early cell cycle progression. We report here a new type of regulation that influences Cdk recognition and phosphorylation of substrate proteins, mediated through the targeted methylation of a critical lysine residue in the Cdk substrate recognition site. In pRb, lysine (K) 810 represents the essential and conserved basic residue (SPXK) required for cyclin/Cdk recognition and phosphorylation. Methylation of K810 by the methyltransferase Set7/9 impedes binding of Cdk and thereby prevents subsequent phosphorylation of the associated serine (S) residue, retaining pRb in the hypophosphorylated growth-suppressing state. Methylation of K810 is under DNA damage control, and methylated K810 impacts on phosphorylation at sites throughout the pRb protein. Set7/9 is required for efficient cell cycle arrest, and significantly, a mutant derivative of pRb that cannot be methylated at K810 exhibits compromised cell cycle arrest. Thus, the regulation of phosphorylation by Cdks reflects the combined interplay with methylation events, and more generally the targeted methylation of a lysine residue within a Cdk-consensus site in pRb represents an important point of control in cell cycle progression. PMID:21119616

  2. Parkinsonism-associated protein DJ-1/Park7 is a major protein deglycase that repairs methylglyoxal- and glyoxal-glycated cysteine, arginine, and lysine residues.

    PubMed

    Richarme, Gilbert; Mihoub, Mouadh; Dairou, Julien; Bui, Linh Chi; Leger, Thibaut; Lamouri, Aazdine

    2015-01-16

    Glycation is an inevitable nonenzymatic covalent reaction between proteins and endogenous reducing sugars or dicarbonyls (methylglyoxal, glyoxal) that results in protein inactivation. DJ-1 was reported to be a multifunctional oxidative stress response protein with poorly defined function. Here, we show that human DJ-1 is a protein deglycase that repairs methylglyoxal- and glyoxal-glycated amino acids and proteins by acting on early glycation intermediates and releases repaired proteins and lactate or glycolate, respectively. DJ-1 deglycates cysteines, arginines, and lysines (the three major glycated amino acids) of serum albumin, glyceraldehyde-3-phosphate dehydrogenase, aldolase, and aspartate aminotransferase and thus reactivates these proteins. DJ-1 prevented protein glycation in an Escherichia coli mutant deficient in the DJ-1 homolog YajL and restored cell viability in glucose-containing media. These results suggest that DJ-1-associated Parkinsonism results from excessive protein glycation and establishes DJ-1 as a major anti-glycation and anti-aging protein. PMID:25416785

  3. Parkinsonism-associated Protein DJ-1/Park7 Is a Major Protein Deglycase That Repairs Methylglyoxal- and Glyoxal-glycated Cysteine, Arginine, and Lysine Residues

    PubMed Central

    Richarme, Gilbert; Mihoub, Mouadh; Dairou, Julien; Bui, Linh Chi; Leger, Thibaut; Lamouri, Aazdine

    2015-01-01

    Glycation is an inevitable nonenzymatic covalent reaction between proteins and endogenous reducing sugars or dicarbonyls (methylglyoxal, glyoxal) that results in protein inactivation. DJ-1 was reported to be a multifunctional oxidative stress response protein with poorly defined function. Here, we show that human DJ-1 is a protein deglycase that repairs methylglyoxal- and glyoxal-glycated amino acids and proteins by acting on early glycation intermediates and releases repaired proteins and lactate or glycolate, respectively. DJ-1 deglycates cysteines, arginines, and lysines (the three major glycated amino acids) of serum albumin, glyceraldehyde-3-phosphate dehydrogenase, aldolase, and aspartate aminotransferase and thus reactivates these proteins. DJ-1 prevented protein glycation in an Escherichia coli mutant deficient in the DJ-1 homolog YajL and restored cell viability in glucose-containing media. These results suggest that DJ-1-associated Parkinsonism results from excessive protein glycation and establishes DJ-1 as a major anti-glycation and anti-aging protein. PMID:25416785

  4. The chemical properties and functional role of a lysine residue within the active site of native sodium and potassium ion-activated adenosinetriphosphatase

    SciTech Connect

    Xu, K.Y.

    1988-01-01

    The peptide, HLLVMKGAPER, which contains Lysine 501 of the {alpha} polypeptide can be released from intact sodium and potassium ion activated adenosinetriphosphatase by tryptic digestion. An immunoadsorbent directed against the carboxy-terminal, -GAPER, has been constructed. Sealed, right-side-out vesicles, prepared from canine renal kidneys, were labeled with pyridoxal phosphate and sodium ({sup 3}H)borohydride in the absence or presence of saponin, respectively. Large increases in the incorporation of radioactivity into the peptides bound by the immunoadsorbent were observed in the digest obtained from the vesicles exposed to saponin. From the results of several control experiments examining the labeling reaction it could be concluded that the increase in the extent of modification was due to the cytoplasmic disposition of this segment in the native enzyme.

  5. Druggability of methyl-lysine binding sites.

    PubMed

    Santiago, C; Nguyen, K; Schapira, M

    2011-12-01

    Structural modules that specifically recognize--or read--methylated or acetylated lysine residues on histone peptides are important components of chromatin-mediated signaling and epigenetic regulation of gene expression. Deregulation of epigenetic mechanisms is associated with disease conditions, and antagonists of acetyl-lysine binding bromodomains are efficacious in animal models of cancer and inflammation, but little is known regarding the druggability of methyl-lysine binding modules. We conducted a systematic structural analysis of readers of methyl marks and derived a predictive druggability landscape of methyl-lysine binding modules. We show that these target classes are generally less druggable than bromodomains, but that some proteins stand as notable exceptions. PMID:22146969

  6. Contamination of Critical Surfaces from NVR Glove Residues Via Dry Handling and Solvent Cleaning

    NASA Technical Reports Server (NTRS)

    Sovinski, Marjorie F.

    2004-01-01

    Gloves are often used to prevent the contamination of critical surfaces during handling. The type of glove chosen for use should be the glove that produces the least amount of non-volatile residue (NVR). This paper covers the analysis of polyethylene, nitrile, latex, vinyl, and polyurethane gloves using the contact transfer and gravimetric determination methods covered in the NASA GSFC work instruction Gravimetric Determination and Contact Transfer of Non-volatile Residue (NVR) in Cleanroom Glove Samples, 541-WI-5330.1.21 and in the ASTM Standard E-1731M-95, Standard Test Method for Gravimetric Determination of Non-Volatile Residue from Cleanroom Gloves. The tests performed focus on contamination of critical surfaces at the molecular level. The study found that for the most part, all of the gloves performed equally well in the contact transfer testing. However, the polyethylene gloves performed the best in the gravimetric determination testing, and therefore should be used whenever solvent contact is a possibility. The nitrile gloves may be used as a substitute for latex gloves when latex sensitivity is an issue. The use of vinyl gloves should be avoided, especially if solvent contact is a possibility. A glove database will be established by Goddard Space Flight Center (GSFC) Code 541 to compile the results from future testing of new gloves and different glove lots.

  7. Critical body residues in the marine amphipod Ampelisca abdita: Sediment exposures with nonionic organic contaminants

    SciTech Connect

    Fay, A.A.; Brownawell, B.J.; Elskus, A.A.; McElroy, A.E.

    2000-04-01

    Body residues associated with acute toxicity were determined in the marine amphipod Ampelisca abdita exposed to spiked sediments. Nonylphenol and 2,2{prime},4,4{prime}-tetrachlorobiphenyl critical body residues (CBRs, body residue of contaminant at 50% mortality) were 1.1 {micro}mol/g wet tissue and 0.57 {micro}mol/g wet tissue, respectively, values near the low end of the CBR range expected for compounds acting via narcosis. The polycyclic aromatic hydrocarbons tested, benzo[a]pyrene (BaP) and benz[a]anthracene (BaA), were not acutely toxic at exposure concentrations of up to 43 and 1,280 {micro}g/g dry sediment for BaA and BaP respectively, and body burdens up to 1.2 {micro}mol/g wet tissue (for BaP). Neither polycyclic aromatic hydrocarbon (PAH) was significantly metabolized by A. abdita. The microextraction technique employed here allowed residue analysis of samples containing as few as three amphipods (0.33 mg dry wt). The CBR approach avoids confounding factors such as variations in bioavailability and uptake kinetics and could be employed to assess the relative contribution of specific contaminants or contaminant classes in mixtures to effects observed in toxicity tests with Ampelisca and other organisms.

  8. Charged/Polar-Residue Scanning of the Hydrophobic Face of Transmembrane Domain 9 of the Yeast Glutathione Transporter, Hgt1p, Reveals a Conformationally Critical Region for Substrate Transport

    PubMed Central

    Thakur, Anil; Bachhawat, Anand K.

    2015-01-01

    Unraveling the mechanistic workings of membrane transporters has remained a challenging task. We describe a novel strategy that involves subjecting the residues of the hydrophobic face of a transmembrane helix to a charged/polar scanning mutagenesis. TMD9 of the yeast glutathione transporter, Hgt1p, has been identified as being important in substrate binding, and two residues, F523 and Q526, are expected to line the substrate translocation channel while the other face is hydrophobic. The hydrophobic face of TMD9 helix consists of residues A509, V513, L517, L520, I524, and I528, and these were mutated to lysine, glutamine, and glutamic acid. Among the 16 charged mutants created, six were nonfunctional, revealing a surprising tolerance of charged residues in the hydrophobic part of TM helices. Furthermore, the only position that did not tolerate any charged residue was I524, proximal to the substrate binding residues. However, P525, also proximal to the substrate binding residues, did tolerate charged/polar residues, suggesting that mere proximity to the substrate binding residues was not the only factor. The I524K/E/Q mutants expressed well and localized correctly despite lacking any glutathione uptake capability. Isolation of suppressors for all nonfunctional mutants yielded second-site suppressors only for I524K and I524Q, and suppressors for these mutations appeared at G202K/I and G202K/Q, respectively. G202 is in the hydrophilic loop between TMD3 and TMD4. The results suggest that I524 in the hydrophobic face interacts with this region and is also in a conformationally critical region for substrate translocation. PMID:25784163

  9. Residual stress and damage-induced critical fracture on CO2 laser treated fused silica

    SciTech Connect

    Matthews, M; Stolken, J; Vignes, R; Norton, M

    2009-11-02

    Localized damage repair and polishing of silica-based optics using mid- and far-IR CO{sub 2} lasers has been shown to be an effective method for increasing optical damage threshold in the UV. However, it is known that CO{sub 2} laser heating of silicate surfaces can lead to a level of residual stress capable of causing critical fracture either during or after laser treatment. Sufficient control of the surface temperature as a function of time and position is therefore required to limit this residual stress to an acceptable level to avoid critical fracture. In this work they present the results of 351 nm, 3 ns Gaussian damage growth experiments within regions of varying residual stress caused by prior CO{sub 2} laser exposures. Thermally stressed regions were non-destructively characterized using polarimetry and confocal Raman microscopy to measure the stress induced birefringence and fictive temperature respectively. For 1 {approx} 40s square pulse CO{sub 2} laser exposures created over 0.5-1.25 kW/cm{sup 2} with a 1-3 mm 1/e{sup 2} diameter beam (T{sub max} {approx} 1500-3000 K), the critical damage site size leading to fracture increases weakly with peak temperature, but shows a stronger dependence on cooling rate, as predicted by finite element hydrodynamics simulations. Confocal micro-Raman was used to probe structural changes to the glass over different thermal histories and indicated a maximum fictive temperature of 1900K for T{sub max} {ge} 2000 K. The effect of cooling rate on fictive temperature caused by CO{sub 2} laser heating are consistent with finite element calculations based on a Tool-Narayanaswamy relaxation model.

  10. Mutation analysis of the Pip interaction domain reveals critical residues for protein–protein interactions

    PubMed Central

    Ortiz, Maria Antonia; Light, James; Maki, Richard A.; Assa-Munt, Nuria

    1999-01-01

    The PU.1 interaction partner (Pip) is a member of the interferon regulatory factor family that regulates gene expression through heterodimerization with the ETS transcription factor PU.1. Binding of Pip alone to DNA is weak, and usually it is recruited by phosphorylated PU.1 to form a strong ternary complex with specific DNA sequences. An approach combining sequence homology analysis, secondary structure predictions, and a precise mutational strategy has been used to determine critical residues within the Pip heterodimerization domain that contribute to ternary complex formation. We have delimited the Pip interaction domain to residues 245–422 by using deletion analysis. Site-directed mutagenesis of conserved polar amino acids within two predicted α-helices contained in this region, and which are highly conserved in the IRF family, confirmed the importance of these residues for Pip–PU.1 interaction with DNA as well as for trans-activation activity. Our results suggest the existence of a functional epitope essential for heterodimerization between Pip and PU.1 and possibly, in general, between interferon regulatory factor family members and their partners. PMID:10077581

  11. Metabolism leaves its mark on the powerhouse: recent progress in post-translational modifications of lysine in mitochondria

    PubMed Central

    Papanicolaou, Kyriakos N.; O'Rourke, Brian; Foster, D. Brian

    2014-01-01

    Lysine modifications have been studied extensively in the nucleus, where they play pivotal roles in gene regulation and constitute one of the pillars of epigenetics. In the cytoplasm, they are critical to proteostasis. However, in the last decade we have also witnessed the emergence of mitochondria as a prime locus for post-translational modification (PTM) of lysine thanks, in large measure, to evolving proteomic techniques. Here, we review recent work on evolving set of PTM that arise from the direct reaction of lysine residues with energized metabolic thioester-coenzyme A intermediates, including acetylation, succinylation, malonylation, and glutarylation. We highlight the evolutionary conservation, kinetics, stoichiometry, and cross-talk between members of this emerging family of PTMs. We examine the impact on target protein function and regulation by mitochondrial sirtuins. Finally, we spotlight work in the heart and cardiac mitochondria, and consider the roles acetylation and other newly-found modifications may play in heart disease. PMID:25228883

  12. Solid residues from Italian municipal solid waste incinerators: A source for "critical" raw materials.

    PubMed

    Funari, Valerio; Braga, Roberto; Bokhari, Syed Nadeem Hussain; Dinelli, Enrico; Meisel, Thomas

    2015-11-01

    The incineration of municipal solid wastes is an important part of the waste management system along with recycling and waste disposal, and the solid residues produced after the thermal process have received attention for environmental concerns and the recovery of valuable metals. This study focuses on the Critical Raw Materials (CRM) content in solid residues from two Italian municipal waste incinerator (MSWI) plants. We sampled untreated bottom ash and fly ash residues, i.e. the two main outputs of common grate-furnace incinerators, and determined their total elemental composition with sensitive analytical techniques such as XRF and ICP-MS. After the removal of a few coarse metallic objects from bottom ashes, the corresponding ICP solutions were obtained using strong digestion methods, to ensure the dissolution of the most refractory components that could host significant amounts of precious metals and CRM. The integration of accurate chemical data with a substance flow analysis, which takes into account the mass balance and uncertainties assessment, indicates that bottom and fly ashes can be considered as a low concentration stream of precious and high-tech metals. The magnesium, copper, antimony and zinc contents are close to the corresponding values of a low-grade ore. The distribution of the elements flow between bottom and fly ash, and within different grain size fractions of bottom ash, is appraised. Most elements are enriched in the bottom ash flow, especially in the fine grained fractions. However, the calculated transfer coefficients indicate that Sb and Zn strongly partition into the fly ashes. The comparison with available studies indicates that the CRM concentrations in the untreated solid residues are comparable with those residues that undergo post-treatment beneficiations, e.g. separation between ferrous and non-ferrous fractions. The suggested separate collection of "fresh" bottom ash, which could be processed for further mineral upgrading, can

  13. Critical body residues for lethal and sublethal effects of membrane narcotics in the midge, Chironomus riparius

    SciTech Connect

    Fisher, S.W.; Hwang, H.; Landrum, P.F.

    1995-12-31

    The concept of the critical body residue (CBR) offers a compelling new way to evaluate hazard posed by persistent contaminants such as PCBs. The authors tested the utility of using CBRs for PCBs in an invertebrate species, Chironomus riparius. Acute toxicity (< 10 d) tests were performed with 2nd instar larvae by adding trace amounts of {sup 14}C-labeled PCBs to unlabeled PCBs in amounts sufficient to generate a dose-responsive mortality curve. Because of the limited water solubility of the PCBs tested, it was not possible to produce a toxic tissue concentration via aqueous exposure. This difficulty was overcome by allowing the midges to feed upon contaminated algae (Chlorella vulgaris) which could be loaded with much higher levels of PCBs than would dissolve in water. CBRs for acute toxicity were measured for 3 PCBs; an average CBR of about 1 mmol/kg was determined. If the midge CBRs are lipid normalized, they fall into the range of values that have previously been determined for vertebrates. The authors also evaluated CBRs in midges for a variety of sublethal impairments including development time within a stadium, larval weight and fecundity. A CBR of 1.09 {micro}mol/kg resulted in a significant increase in larval development time and decrease in larval weight for second instar larvae. Tissue residues declined in the third and fourth instars, despite continuing exposure suggesting that the animals developed attenuating mechanisms or that contaminant loss at ecdysis is significant. Despite declining tissue residues throughout the larval instars, fecundity was reduced from 284 eggs/female in controls to 244 eggs/female in animals exposed to the highest sublethal concentration.

  14. Bridge helix bending promotes RNA polymerase II backtracking through a critical and conserved threonine residue

    NASA Astrophysics Data System (ADS)

    da, Lin-Tai; Pardo-Avila, Fátima; Xu, Liang; Silva, Daniel-Adriano; Zhang, Lu; Gao, Xin; Wang, Dong; Huang, Xuhui

    2016-04-01

    The dynamics of the RNA polymerase II (Pol II) backtracking process is poorly understood. We built a Markov State Model from extensive molecular dynamics simulations to identify metastable intermediate states and the dynamics of backtracking at atomistic detail. Our results reveal that Pol II backtracking occurs in a stepwise mode where two intermediate states are involved. We find that the continuous bending motion of the Bridge helix (BH) serves as a critical checkpoint, using the highly conserved BH residue T831 as a sensing probe for the 3'-terminal base paring of RNA:DNA hybrid. If the base pair is mismatched, BH bending can promote the RNA 3'-end nucleotide into a frayed state that further leads to the backtracked state. These computational observations are validated by site-directed mutagenesis and transcript cleavage assays, and provide insights into the key factors that regulate the preferences of the backward translocation.

  15. Bridge helix bending promotes RNA polymerase II backtracking through a critical and conserved threonine residue

    PubMed Central

    Da, Lin-Tai; Pardo-Avila, Fátima; Xu, Liang; Silva, Daniel-Adriano; Zhang, Lu; Gao, Xin; Wang, Dong; Huang, Xuhui

    2016-01-01

    The dynamics of the RNA polymerase II (Pol II) backtracking process is poorly understood. We built a Markov State Model from extensive molecular dynamics simulations to identify metastable intermediate states and the dynamics of backtracking at atomistic detail. Our results reveal that Pol II backtracking occurs in a stepwise mode where two intermediate states are involved. We find that the continuous bending motion of the Bridge helix (BH) serves as a critical checkpoint, using the highly conserved BH residue T831 as a sensing probe for the 3′-terminal base paring of RNA:DNA hybrid. If the base pair is mismatched, BH bending can promote the RNA 3′-end nucleotide into a frayed state that further leads to the backtracked state. These computational observations are validated by site-directed mutagenesis and transcript cleavage assays, and provide insights into the key factors that regulate the preferences of the backward translocation. PMID:27091704

  16. Charge Neutralization of the Central Lysine Cluster in Prion Protein (PrP) Promotes PrPSc-like Folding of Recombinant PrP Amyloids*

    PubMed Central

    Groveman, Bradley R.; Kraus, Allison; Raymond, Lynne D.; Dolan, Michael A.; Anson, Kelsie J.; Dorward, David W.; Caughey, Byron

    2015-01-01

    The structure of the infectious form of prion protein, PrPSc, remains unclear. Most pure recombinant prion protein (PrP) amyloids generated in vitro are not infectious and lack the extent of the protease-resistant core and solvent exclusion of infectious PrPSc, especially within residues ∼90–160. Polyanionic cofactors can enhance infectivity and PrPSc-like characteristics of such fibrils, but the mechanism of this enhancement is unknown. In considering structural models of PrPSc multimers, we identified an obstacle to tight packing that might be overcome with polyanionic cofactors, namely, electrostatic repulsion between four closely spaced cationic lysines within a central lysine cluster of residues 101–110. For example, in our parallel in-register intermolecular β-sheet model of PrPSc, not only would these lysines be clustered within the 101–110 region of the primary sequence, but they would have intermolecular spacings of only ∼4.8 Å between stacked β-strands. We have now performed molecular dynamics simulations predicting that neutralization of the charges on these lysine residues would allow more stable parallel in-register packing in this region. We also show empirically that substitution of these clustered lysine residues with alanines or asparagines results in recombinant PrP amyloid fibrils with extended proteinase-K resistant β-sheet cores and infrared spectra that are more reminiscent of bona fide PrPSc. These findings indicate that charge neutralization at the central lysine cluster is critical for the folding and tight packing of N-proximal residues within PrP amyloid fibrils. This charge neutralization may be a key aspect of the mechanism by which anionic cofactors promote PrPSc formation. PMID:25416779

  17. Positive charges on lysine residues of the extrinsic 18 kDa protein are important to its electrostatic interaction with spinach photosystem II membranes.

    PubMed

    Gao, Jin-Peng; Yong, Zhen-Hua; Zhang, Feng; Ruan, Kang-Cheng; Xu, Chun-He; Chen, Gen-Yun

    2005-11-01

    To determine the contribution of charged amino acids to binding with the photosystem II complex (PSII), the amino or carboxyl groups of the extrinsic 18 kDa protein were modified with N-succinimidyl propionate (NSP) or glycine methyl ester (GME) in the presence of a water-soluble carbodiimide, respectively. Based on isoelectric point shift, 4-10 and 10-14 amino groups were modified in the presence of 2 and 4 mM NSP, respectively. Similarly, 3-4 carboxyl groups were modified by reaction with 100 mM GME. Neutralization of negatively charged carboxyl groups with GME did not alter the binding activity of the extrinsic 18 kDa protein. However, the NSP-modified 18 kDa protein, in which the positively charged amino groups had been modified to uncharged methyl esters, failed to bind with the PSII membrane in the presence of the extrinsic 23 kDa protein. This defect can not be attributed to structural or conformational alterations imposed by chemical modification, as the fluorescence and circular dichroism spectra among native, GME- and NSP-modified extrinsic 18 kDa proteins were similar. Thus, we have concluded that the positive charges of lysyl residues in the extrinsic 18 kDa protein are important for its interaction with PSII membranes in the presence of the extrinsic 23 kDa protein. Furthermore, it was found that the negative charges of carboxyl groups of this protein did not participate in binding with the extrinsic 23 kDa protein associated with PSII membranes. PMID:16270152

  18. A Method to Determine Lysine Acetylation Stoichiometries

    DOE PAGESBeta

    Nakayasu, Ernesto S.; Wu, Si; Sydor, Michael A.; Shukla, Anil K.; Weitz, Karl K.; Moore, Ronald J.; Hixson, Kim K.; Kim, Jong-Seo; Petyuk, Vladislav A.; Monroe, Matthew E.; et al

    2014-01-01

    Lysine acetylation is a common protein posttranslational modification that regulates a variety of biological processes. A major bottleneck to fully understanding the functional aspects of lysine acetylation is the difficulty in measuring the proportion of lysine residues that are acetylated. Here we describe a mass spectrometry method using a combination of isotope labeling and detection of a diagnostic fragment ion to determine the stoichiometry of protein lysine acetylation. Using this technique, we determined the modification occupancy for ~750 acetylated peptides from mammalian cell lysates. Furthermore, the acetylation on N-terminal tail of histone H4 was cross-validated by treating cells with sodiummore » butyrate, a potent deacetylase inhibitor, and comparing changes in stoichiometry levels measured by our method with immunoblotting measurements. Of note we observe that acetylation stoichiometry is high in nuclear proteins, but very low in mitochondrial and cytosolic proteins. In summary, our method opens new opportunities to study in detail the relationship of lysine acetylation levels of proteins with their biological functions.« less

  19. Saccharomyces cerevisiae a-Factor Mutants Reveal Residues Critical for Processing, Activity, and Export

    PubMed Central

    Huyer, Gregory; Kistler, Amy; Nouvet, Franklin J.; George, Carolyn M.; Boyle, Meredith L.; Michaelis, Susan

    2006-01-01

    The Saccharomyces cerevisiae mating pheromone a-factor provides a paradigm for understanding the biogenesis of prenylated fungal pheromones. The biogenesis of a-factor involves multiple steps: (i) C-terminal CAAX modification (where C is cysteine, A is aliphatic, and X is any residue) which includes prenylation, proteolysis, and carboxymethylation (by Ram1p/Ram2p, Ste24p or Rce1p, and Ste14p, respectively); (ii) N-terminal processing, involving two sequential proteolytic cleavages (by Ste24p and Axl1p); and (iii) nonclassical export (by Ste6p). Once exported, mature a-factor interacts with the Ste3p receptor on MATα cells to stimulate mating. The a-factor biogenesis machinery is well defined, as is the CAAX motif that directs C-terminal modification; however, very little is known about the sequence determinants within a-factor required for N-terminal processing, activity, and export. Here we generated a large collection of a-factor mutants and identified residues critical for the N-terminal processing steps mediated by Ste24p and Axl1p. We also identified mutants that fail to support mating but do not affect biogenesis or export, suggesting a defective interaction with the Ste3p receptor. Mutants significantly impaired in export were also found, providing evidence that the Ste6p transporter recognizes sequence determinants as well as CAAX modifications. We also performed a phenotypic analysis of the entire set of isogenic a-factor biogenesis machinery mutants, which revealed information about the dependency of biogenesis steps upon one another, and demonstrated that export by Ste6p requires the completion of all processing events. Overall, this comprehensive analysis will provide a useful framework for the study of other fungal pheromones, as well as prenylated metazoan proteins involved in development and aging. PMID:16963638

  20. A chemical proteomics approach for global analysis of lysine monomethylome profiling.

    PubMed

    Wu, Zhixiang; Cheng, Zhongyi; Sun, Mingwei; Wan, Xuelian; Liu, Ping; He, Tieming; Tan, Minjia; Zhao, Yingming

    2015-02-01

    Methylation of lysine residues on histone proteins is known to play an important role in chromatin structure and function. However, non-histone protein substrates of this modification remain largely unknown. An effective approach for system-wide analysis of protein lysine methylation, particularly lysine monomethylation, is lacking. Here we describe a chemical proteomics approach for global screening for monomethyllysine substrates, involving chemical propionylation of monomethylated lysine, affinity enrichment of the modified monomethylated peptides, and HPLC/MS/MS analysis. Using this approach, we identified with high confidence 446 lysine monomethylation sites in 398 proteins, including three previously unknown histone monomethylation marks, representing the largest data set of protein lysine monomethylation described to date. Our data not only confirms previously discovered lysine methylation substrates in the nucleus and spliceosome, but also reveals new substrates associated with diverse biological processes. This method hence offers a powerful approach for dynamic study of protein lysine monomethylation under diverse cellular conditions and in human diseases. PMID:25505155

  1. Critical role of lysine 134 methylation on histone H2AX for γ-H2AX production and DNA repair

    PubMed Central

    Sone, Kenbun; Piao, Lianhua; Nakakido, Makoto; Ueda, Koji; Jenuwein, Thomas; Nakamura, Yusuke; Hamamoto, Ryuji

    2014-01-01

    The presence of phosphorylated histone H2AX (γ-H2AX) is associated with the local activation of DNA-damage repair pathways. Although γ-H2AX deregulation in cancer has previously been reported, the molecular mechanism involved and its relationship with other histone modifications remain largely unknown. Here we find that the histone methyltransferase SUV39H2 methylates histone H2AX on lysine 134. When H2AX was mutated to abolish K134 methylation, the level of γ-H2AX became significantly reduced. We also found lower γ-H2AX activity following the introduction of double-strand breaks in Suv39h2 knockout cells or on SUV39H2 knockdown. Tissue microarray analyses of clinical lung and bladder tissues also revealed a positive correlation between H2AX K134 methylation and γ-H2AX levels. Furthermore, introduction of K134-substituted histone H2AX enhanced radio- and chemosensitivity of cancer cells. Overall, our results suggest that H2AX methylation plays a role in the regulation of γ-H2AX abundance in cancer. PMID:25487737

  2. Selective cleavage enhanced by acetylating the side chain of lysine.

    PubMed

    Fu, Leixiaomeng; Chen, Tingting; Xue, Gaiqing; Zu, Lily; Fang, Weihai

    2013-01-01

    Selective cleavage is of great interest in mass spectrometry studies as it can help sequence identification by promoting simple fragmentation pattern of peptides and proteins. In this work, the collision-induced dissociation of peptides containing internal lysine and acetylated lysine residues were studied. The experimental and computational results revealed that multiple fragmentation pathways coexisted when the lysine residue was two amino acid residues away from N-terminal of the peptide. After acetylation of the lysine side-chain, b(n)+ ions were the most abundant primary fragment products and the Lys(Ac)-Gly amide bond became the dominant cleavage site via an oxazolone pathway. Acetylating the side-chain of lysine promoted the selective cleavage of Lys-Xxx amide bond and generated much more information of the peptide backbone sequence. The results re-evaluate the selective cleavage due to the lysine basic side-chain and provide information for studying the post-translational modification of proteins and other bio-molecules containing Lys residues. PMID:23303756

  3. Mutagenesis identifies the critical amino acid residues of human endonuclease G involved in catalysis, magnesium coordination, and substrate specificity

    PubMed Central

    Wu, Shih-Lu; Li, Chia-Cheng; Chen, Jaw-Chyun; Chen, Yi-Jin; Lin, Ching-Ting; Ho, Tin-Yun; Hsiang, Chien-Yun

    2009-01-01

    Background Endonuclease G (EndoG), a member of DNA/RNA nonspecific ββα-Me-finger nucleases, is involved in apoptosis and normal cellular proliferation. In this study, we analyzed the critical amino acid residues of EndoG and proposed the catalytic mechanism of EndoG. Methods To identify the critical amino acid residues of human EndoG, we replaced the conserved histidine, asparagine, and arginine residues with alanine. The catalytic efficacies of Escherichia coli-expressed EndoG variants were further analyzed by kinetic studies. Results Diethyl pyrocarbonate modification assay revealed that histidine residues were involved in EndoG activity. His-141, Asn-163, and Asn-172 in the H-N-H motif of EndoG were critical for catalysis and substrate specificity. H141A mutant required a higher magnesium concentration to achieve its activity, suggesting the unique role of His-141 in both catalysis and magnesium coordination. Furthermore, an additional catalytic residue (Asn-251) and an additional metal ion binding site (Glu-271) of human EndoG were identified. Conclusion Based on the mutational analysis and homology modeling, we proposed that human EndoG shared a similar catalytic mechanism with nuclease A from Anabaena. PMID:19272175

  4. Probing the critical residues for intramolecular fructosyl transfer reaction of a levan fructotransferase.

    PubMed

    Moon, Keum-Ok; Choi, Kyoung-Hwa; Kang, Ho-Young; Oh, Jeong-Il; Jang, Se Bok; Park, Cheon-Seok; Lee, Jong-Hoon; Cha, Jaeho

    2008-06-01

    Levan fructotransferase (LFTase) preferentially catalyzes the transfructosylation reaction in addition to levan hydrolysis, whereas other levan-degrading enzymes hydrolyze levan into a levan-oligosaccharide and fructose. Based on sequence comparisons and enzymatic properties, the fructosyl transfer activity of LFTase is proposed to have evolved from levanase. In order to probe the residues that are critical to the intramolecular fructosyl transfer reaction of the Microbacterium sp. AL-210 LFTase, an error-prone PCR mutagenesis process was carried out, and the mutants that led to a shift in activity from transfructosylation towards hydrolysis of levan were screened by the DNS method. After two rounds of mutagenesis, TLC and HPLC analyses of the reaction products by the selected mutants revealed two major products; one is a di-D-fructose- 2,6':6,2'-dianhydride (DFAIV) and the other is a levanbiose. The newly detected levanbiose corresponds to the reaction product from LFTase lacking transferring activity. Two mutants (2-F8 and 2-G9) showed a high yield of levanbiose (38-40%) compared with the wild-type enzyme, and thus behaved as levanases. Sequence analysis of the individual mutants responsible for the enhanced hydrolytic activity indicated that Asn-85 was highly involved in the transfructosylation activity of LFTase. PMID:18600048

  5. Residues in the hendra virus fusion protein transmembrane domain are critical for endocytic recycling.

    PubMed

    Popa, Andreea; Carter, James R; Smith, Stacy E; Hellman, Lance; Fried, Michael G; Dutch, Rebecca Ellis

    2012-03-01

    Hendra virus is a highly pathogenic paramyxovirus classified as a biosafety level four agent. The fusion (F) protein of Hendra virus is critical for promoting viral entry and cell-to-cell fusion. To be fusogenically active, Hendra virus F must undergo endocytic recycling and cleavage by the endosomal/lysosomal protease cathepsin L, but the route of Hendra virus F following internalization and the recycling signals involved are poorly understood. We examined the intracellular distribution of Hendra virus F following endocytosis and showed that it is primarily present in Rab5- and Rab4-positive endosomal compartments, suggesting that cathepsin L cleavage occurs in early endosomes. Hendra virus F transmembrane domain (TMD) residues S490 and Y498 were found to be important for correct Hendra virus F recycling, with the hydroxyl group of S490 and the aromatic ring of Y498 important for this process. In addition, changes in association of isolated Hendra virus F TMDs correlated with alterations to Hendra virus F recycling, suggesting that appropriate TMD interactions play an important role in endocytic trafficking. PMID:22238299

  6. Canine Distemper Virus Fusion Activation: Critical Role of Residue E123 of CD150/SLAM

    PubMed Central

    Khosravi, Mojtaba; Bringolf, Fanny; Röthlisberger, Silvan; Bieringer, Maria; Schneider-Schaulies, Jürgen; Zurbriggen, Andreas; Origgi, Francesco

    2015-01-01

    ABSTRACT Measles virus (MeV) and canine distemper virus (CDV) possess tetrameric attachment proteins (H) and trimeric fusion proteins, which cooperate with either SLAM or nectin 4 receptors to trigger membrane fusion for cell entry. While the MeV H-SLAM cocrystal structure revealed the binding interface, two distinct oligomeric H assemblies were also determined. In one of the conformations, two SLAM units were sandwiched between two discrete H head domains, thus spotlighting two binding interfaces (“front” and “back”). Here, we investigated the functional relevance of both interfaces in activating the CDV membrane fusion machinery. While alanine-scanning mutagenesis identified five critical regulatory residues in the front H-binding site of SLAM, the replacement of a conserved glutamate residue (E at position 123, replaced with A [E123A]) led to the most pronounced impact on fusion promotion. Intriguingly, while determination of the interaction of H with the receptor using soluble constructs revealed reduced binding for the identified SLAM mutants, no effect was recorded when physical interaction was investigated with the full-length counterparts of both molecules. Conversely, although mutagenesis of three strategically selected residues within the back H-binding site of SLAM did not substantially affect fusion triggering, nevertheless, the mutants weakened the H-SLAM interaction recorded with the membrane-anchored protein constructs. Collectively, our findings support a mode of binding between the attachment protein and the V domain of SLAM that is common to all morbilliviruses and suggest a major role of the SLAM residue E123, located at the front H-binding site, in triggering the fusion machinery. However, our data additionally support the hypothesis that other microdomain(s) of both glycoproteins (including the back H-binding site) might be required to achieve fully productive H-SLAM interactions. IMPORTANCE A complete understanding of the measles virus

  7. CRITICAL BODY RESIDUES FOR FRESHWATER AND SALTWATER AMPHIPODS EXPOSED TO SEDIMENT CONTAINING A MIXTURE OF HIGH KOW PAHS

    EPA Science Inventory

    Sediments were spiked with a mixture of 13 high log Kow (5.4-6.8) PAH compounds to determine critical body residues (CBR) in Hyalella azteca and Leptocheirus plumulosus. Hyalella were exposed for 28 d in a intermittent flow test and for 10 d in a static test to compare PAH uptake...

  8. Synthesis of Lysine Methyltransferase Inhibitors

    NASA Astrophysics Data System (ADS)

    Ye, Tao; Hui, Chunngai

    2015-07-01

    Lysine methyltransferase which catalyze methylation of histone and nonhistone proteins, play a crucial role in diverse biological processes and has emerged as a promising target for the development of various human diseases, including cancer, inflammation, and psychiatric disorders. However, inhibiting Lysine methyltransferases selectively has presented many challenges to medicinal chemists. During the past decade, lysine methyltransferase inhibitors covering many different structural classes have been designed and developed. In this review, we describe the development of selective, small-molecule inhibitors of lysine methyltransferases with an emphasis on their discovery and chemical synthesis. We highlight the current state of lysine methyltransferase inhibitors and discuss future directions and opportunities for lysine methyltransferase inhibitor discovery.

  9. Cyclic AMP-dependent Protein Lysine Acylation in Mycobacteria Regulates Fatty Acid and Propionate Metabolism*

    PubMed Central

    Nambi, Subhalaxmi; Gupta, Kallol; Bhattacharyya, Moitrayee; Ramakrishnan, Parvathy; Ravikumar, Vaishnavi; Siddiqui, Nida; Thomas, Ann Terene; Visweswariah, Sandhya S.

    2013-01-01

    Acetylation of lysine residues is a posttranslational modification that is used by both eukaryotes and prokaryotes to regulate a variety of biological processes. Here we identify multiple substrates for the cAMP-dependent protein lysine acetyltransferase from Mycobacterium tuberculosis (KATmt). We demonstrate that a catalytically important lysine residue in a number of FadD (fatty acyl CoA synthetase) enzymes is acetylated by KATmt in a cAMP-dependent manner and that acetylation inhibits the activity of FadD enzymes. A sirtuin-like enzyme can deacetylate multiple FadDs, thus completing the regulatory cycle. Using a strain deleted for the KATmt ortholog in Mycobacterium bovis Bacillus Calmette-Guérin (BCG), we show for the first time that acetylation is dependent on intracellular cAMP levels. KATmt can utilize propionyl CoA as a substrate and, therefore, plays a critical role in alleviating propionyl CoA toxicity in mycobacteria by inactivating acyl CoA synthetase (ACS). The precision by which mycobacteria can regulate the metabolism of fatty acids in a cAMP-dependent manner appears to be unparalleled in other biological organisms and is ideally suited to adapt to the complex environment that pathogenic mycobacteria experience in the host. PMID:23553634

  10. SIRT5 regulates the mitochondrial lysine succinylome and metabolic networks.

    PubMed

    Rardin, Matthew J; He, Wenjuan; Nishida, Yuya; Newman, John C; Carrico, Chris; Danielson, Steven R; Guo, Ailan; Gut, Philipp; Sahu, Alexandria K; Li, Biao; Uppala, Radha; Fitch, Mark; Riiff, Timothy; Zhu, Lei; Zhou, Jing; Mulhern, Daniel; Stevens, Robert D; Ilkayeva, Olga R; Newgard, Christopher B; Jacobson, Matthew P; Hellerstein, Marc; Goetzman, Eric S; Gibson, Bradford W; Verdin, Eric

    2013-12-01

    Reversible posttranslational modifications are emerging as critical regulators of mitochondrial proteins and metabolism. Here, we use a label-free quantitative proteomic approach to characterize the lysine succinylome in liver mitochondria and its regulation by the desuccinylase SIRT5. A total of 1,190 unique sites were identified as succinylated, and 386 sites across 140 proteins representing several metabolic pathways including β-oxidation and ketogenesis were significantly hypersuccinylated in Sirt5(-/-) animals. Loss of SIRT5 leads to accumulation of medium- and long-chain acylcarnitines and decreased β-hydroxybutyrate production in vivo. In addition, we demonstrate that SIRT5 regulates succinylation of the rate-limiting ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) both in vivo and in vitro. Finally, mutation of hypersuccinylated residues K83 and K310 on HMGCS2 to glutamic acid strongly inhibits enzymatic activity. Taken together, these findings establish SIRT5 as a global regulator of lysine succinylation in mitochondria and present a mechanism for inhibition of ketogenesis through HMGCS2. PMID:24315375

  11. SIRT5 regulates the mitochondrial lysine succinylome and metabolic networks

    PubMed Central

    Rardin, Matthew J.; He, Wenjuan; Nishida, Yuya; Newman, John C.; Carrico, Chris; Danielson, Steven R.; Guo, Ailan; Gut, Philipp; Sahu, Alexandria K.; Li, Biao; Uppala, Radha; Fitch, Mark; Riiff, Timothy; Zhu, Lei; Zhou, Jing; Mulhern, Daniel; Stevens, Robert D.; Ilkayeva, Olga R.; Newgard, Christopher B.; Jacobson, Matthew P.; Hellerstein, Marc; Goetzman, Eric S.; Gibson, Bradford W.; Verdin, Eric

    2014-01-01

    Summary Reversible posttranslational modifications are emerging as critical regulators of mitochondrial proteins and metabolism. Here, we use a label-free quantitative proteomic approach to characterize the lysine succinylome in liver mitochondria and its regulation by the desuccinylase SIRT5. A total of 1190 unique sites were identified as succinylated, and 386 sites across 140 proteins representing several metabolic pathways including β-oxidation and ketogenesis were significantly hypersuccinylated in Sirt5−/− animals. Loss of SIRT5 leads to accumulation of medium- and long-chain acylcarnitines and decreased β-hydroxybutyrate production in vivo. In addition, we demonstrate that SIRT5 regulates succinylation of the rate-limiting ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) both in vivo and in vitro. Finally, mutation of hypersuccinylated residues K83 and K310 on HMGCS2 to glutamic acid strongly inhibits enzymatic activity. Taken together, these findings establish SIRT5 as a global regulator of lysine succinylation in mitochondria and present a mechanism for inhibition of ketogenesis through HMGCS2. PMID:24315375

  12. Sub-critical water extraction of residual green tea to produce a roasted green tea-like extract.

    PubMed

    Etoh, Hideo; Ohtaki, Naoko; Kato, Hideaki; Kulkarni, Aditya; Morita, Akio

    2010-01-01

    In this study, we investigated the effects of various temperatures between 140 to 260 degrees C during sub-critical water extraction of residual green tea in making a roasted green tea-like extract, a popular beverage in Japan. Each residual green tea extract was analyzed for sensory properties and antioxidative activities, and this revealed 140 degrees C to be the best extraction temperature. 5-Hydroxymethyl-2-furaldehyde, (+)-catechin, and (-)-epicatechin were found to be the major antioxidative compounds in the 140 degrees C extract, along with theanine and some important amino acids. PMID:20378975

  13. Quantitative Profiling of the Activity of Protein Lysine Methyltransferase SMYD2 Using SILAC-Based Proteomics.

    PubMed

    Olsen, Jonathan B; Cao, Xing-Jun; Han, Bomie; Chen, Lisa Hong; Horvath, Alexander; Richardson, Timothy I; Campbell, Robert M; Garcia, Benjamin A; Nguyen, Hannah

    2016-03-01

    The significance of non-histone lysine methylation in cell biology and human disease is an emerging area of research exploration. The development of small molecule inhibitors that selectively and potently target enzymes that catalyze the addition of methyl-groups to lysine residues, such as the protein lysine mono-methyltransferase SMYD2, is an active area of drug discovery. Critical to the accurate assessment of biological function is the ability to identify target enzyme substrates and to define enzyme substrate specificity within the context of the cell. Here, using stable isotopic labeling with amino acids in cell culture (SILAC) coupled with immunoaffinity enrichment of mono-methyl-lysine (Kme1) peptides and mass spectrometry, we report a comprehensive, large-scale proteomic study of lysine mono-methylation, comprising a total of 1032 Kme1 sites in esophageal squamous cell carcinoma (ESCC) cells and 1861 Kme1 sites in ESCC cells overexpressing SMYD2. Among these Kme1 sites is a subset of 35 found to be potently down-regulated by both shRNA-mediated knockdown of SMYD2 and LLY-507, a selective small molecule inhibitor of SMYD2. In addition, we report specific protein sequence motifs enriched in Kme1 sites that are directly regulated by endogenous SMYD2 activity, revealing that SMYD2 substrate specificity is more diverse than expected. We further show direct activity of SMYD2 toward BTF3-K2, PDAP1-K126 as well as numerous sites within the repetitive units of two unique and exceptionally large proteins, AHNAK and AHNAK2. Collectively, our findings provide quantitative insights into the cellular activity and substrate recognition of SMYD2 as well as the global landscape and regulation of protein mono-methylation. PMID:26750096

  14. Histone Lysine Methylation Dynamics: Establishment, Regulation, and Biological Impact

    PubMed Central

    Black, Joshua C.; Van Rechem, Capucine; Whetstine, Johnathan R.

    2013-01-01

    Histone lysine methylation has emerged as a critical player in the regulation of gene expression, cell cycle, genome stability, and nuclear architecture. Over the past decade, a tremendous amount of progress has led to the characterization of methyl modifications and the lysine methyltransferases (KMTs) and lysine demethylases (KDMs) that regulate them. Here, we review the discovery and characterization of the KMTs and KDMs and the methyl modifications they regulate. We discuss the localization of the KMTs and KDMs as well as the distribution of lysine methylation throughout the genome. We highlight how these data have shaped our view of lysine methylation as a key determinant of complex chromatin states. Finally, we discuss the regulation of KMTs and KDMs by proteasomal degradation, posttranscriptional mechanisms, and metabolic status. We propose key questions for the field and highlight areas that we predict will yield exciting discoveries in the years to come. PMID:23200123

  15. A lysine-to-arginine mutation on NEDD8 markedly reduces the activity of cullin RING E3 ligase through the impairment of neddylation cascades

    SciTech Connect

    Sui, Yiyan; Liu, Yaobin; Xu, Guoqiang

    2015-06-12

    Neural-precursor-cell-expressed developmentally down-regulated 8 (NEDD8) is a ubiquitin-like modifier, which forms covalent conjugates on lysines of its substrates. This post-translational modification, neddylation, plays important roles in tumor cell proliferation and viability. Ubiquitin can form diverse polyubiquitin chains, on its seven lysines, which play important functions in various biological processes. However, the roles of lysines in NEDD8 have not been explored. Here, we generated nine NEDD8 point mutants, each with one lysine replaced by an arginine, to study the putative function of lysines in NEDD8. Our experiments discover that Lys27 in NEDD8 is a critical residue for protein neddylation. Replacement of this residue with arginine almost completely eliminates the conjugation of NEDD8 to its substrates. Furthermore, we find that the K27R mutant impairs NEDD8 conjugation to the E2 enzyme, which normally forms thioester bonds for further transferring NEDD8 to its ligases and substrates. Therefore, this mutation completely inhibits global protein neddylation, including neddylation of cullin family proteins, resulting in decreased activity of cullin-RING E3 ligases. This work sheds new light on the roles of NEDD8 lysines on neddylation cascades and provides a dominant negative mutant for the study of neddylation and its biological functions. - Highlights: • Lys27 in NEDD8 is critical for protein neddylation. • NEDD8 K27R mutant impairs the NEDD8 conjugation. • NEDD8 K27R mutant significantly reduces the activity of cullin-RING E3 ligases.

  16. L-lysine fermentation.

    PubMed

    Anastassiadis, Savas

    2007-01-01

    Amino acids are the basic bioelements of proteins, which are the most important macromolecules for the functions of humans and animals. Out of the 20 L-amino acids, ecumenically found in most of living organisms, L-lysine is one of the 9 amino acids which are essential for human and animal nutrition. L-lysine is useful as medicament, chemical agent, food material (food industry) and feed additive (animal food). Its demand has been steadily increasing in recent years and several hundred thousands tones of L-lysine (about 800,000 tones/year) are annually produced worldwide almost by microbial fermentation. The stereospecificity of amino acids (the L isomer) makes the fermentation advantageous compared with synthetic processes. Mutant auxotrophic or resistant to certain chemicals strains of so-called gram positive coryneform bacteria are generally used, including the genera Brevibacterium and Corynebacterium, united to the genus. The significance of Research and Development increased rapidly since the discovery of fermentative amino acid production in the fifties (S. Kinoshita et al., Proceedings of the International Symposium on Enzyme Chemistry 2:464-468 (1957)), leading to innovative fermentation processes which replaced the classical manufacturing methods of L-lysine like acid hydrolysis. L-Lysine is separated and purified by suitable downstream processes involving classical separation or extraction methods (ultrafiltration or centrifugation, separation or ion exchange extraction, crystallization, drying) and is sold as a powder. Alternatively, spray dried pellets or liquid fermentation broth can be used as animal feed supplement. On behalf of today's strong competition in amino acid industry, Biotechnology companies are continuously aiming in innovative research developments and use complex management concepts and business strategies, towards gaining market leadership in the field of amino acid production. PMID:19075830

  17. Critical review on the withdrawal period calculation for injection site residues.

    PubMed

    Sanquer, A; Wackowiez, G; Havrileck, B

    2006-10-01

    This review concerns a statistical method for calculating withdrawal period for injection site residues. A recently adopted Committee for Medicinal Products for Veterinary Use/European Agency for the Evaluation of Medicinal Product (CVMP/EMEA) guideline recommends to apply the same method for the calculation of withdrawal period for injection site residues as for other edible tissues. For reasons in this study developed below, this approach is deemed to be inappropriate for the injection site residues. The injection site residues often violate regression assumptions with regard to homoscedasticity (same variance in residue concentrations for different slaughter times) and linearity (of the mean depletion curve in log(e)-scale). The currently recommended method cannot adequately handle these aspects. An alternative pragmatic method taking into account the last slaughter time with all data below the reference threshold, combined with a safety span, is proposed for injection site residues. A nonparametric approach for calculating the withdrawal period is also presumed to be a sound alternative. The references commonly used are the Maximum Residue Limit (MRL) and the Acceptable Daily Intake (ADI). Unfortunately these references are not relevant to the acute risk exposure associated with injection site consumption. The use of alternative references, such as the Acceptable Single Dose Intake (ASDI) or the Acute Reference Dose (ARD) are thought to be more appropriate. PMID:16958779

  18. Differential acute toxicity of tetrachlorobenzene isomers to oligochaetes in soil and water: application of the critical body residue concept.

    PubMed

    Hurdzan, Christopher M; Lanno, Roman P; Sovic, David M

    2011-09-01

    The acute, lethal potency of the 1,2,3,4-, 1,2,4,5- and 1,2,3,5-tetrachlorobenzene isomers was compared in the terrestrial and aquatic oligochaetes Eisenia andrei and Tubifex tubifex. 1,2,4,5-TeCB was neither lethal, nor produced any perceptible adverse effects, at lipid normalized concentrations predicted to be lethal according to the well-established critical body residue concept. If a narcotic is defined as a substance capable of inducing narcosis, rather than a substance displaying certain physical or chemical properties (e.g., log K(ow)), then we do not believe these findings challenge the critical body residue because by the former definition, 1,2,4,5-tetrachlorobenzene is not a narcotic. PMID:21688121

  19. Residue Val237 is critical for the enantioselectivity of Penicillium expansum lipase.

    PubMed

    Tang, Lianghua; Su, Min; Chi, Liying; Zhang, Junling; Zhang, Huihui; Zhu, Ling

    2014-03-01

    The shape of the hydrophobic tunnel leading to the active site of Penicillium expansum lipase (PEL) was redesigned by single-point mutations, in order to better understand enzyme enantioselectivity towards naproxen. A variant with a valine-to-glycine substitution at residue 237 exhibited almost no enantioselectivity (E = 1.1) compared with that (E = 104) of wild-type PEL. The function of the residue, Val237, in the hydrophobic tunnel was further analyzed by site-directed mutagenesis. For each of these variants a significant decrease of enantioselectivity (E < 7) was observed compared with that of wild-type enzyme. Further docking result showed that Val237 plays the most important role in stabilizing the correct orientation of (R)-naproxen. Overall, these results indicate that the residue Val237 is the key amino acid residue maintaining the enantioselectivity of the lipase. PMID:24338160

  20. Functional role of putative critical residues in Mycobacterium tuberculosis RNase P protein.

    PubMed

    Singh, Alla; Ubaid-Ullah, Shah; Batra, Janendra K

    2016-09-01

    RNase P is involved in processing the 5' end of pre-tRNA molecules. Bacterial RNase P contains a catalytic RNA subunit and a protein subunit. In this study, we have analyzed the residues in RNase P protein of M. tuberculosis that differ from the residues generally conserved in other bacterial RNase Ps. The residues investigated in the current study include the unique residues, Val27, Ala70, Arg72, Ala77, and Asp124, and also Phe23 and Arg93 which have been found to be important in the function of RNase P protein components of other bacteria. The selected residues were individually mutated either to those present in other bacterial RNase P protein components at respective positions or in some cases to alanine. The wild type and mutant M. tuberculosis RNase P proteins were expressed in E. coli, purified, used to reconstitute holoenzymes with wild type RNA component in vitro, and functionally characterized. The Phe23Ala and Arg93Ala mutants showed very poor catalytic activity when reconstituted with the RNA component. The catalytic activity of holoenzyme with Val27Phe, Ala70Lys, Arg72Leu and Arg72Ala was also significantly reduced, whereas with Ala77Phe and Asp124Ser the activity of holoenzyme was similar to that with the wild type protein. Although the mutants did not suffer from any binding defects, Val27Phe, Ala70Lys, Arg72Ala and Asp124Ser were less tolerant towards higher temperatures as compared to the wild type protein. The Km of Val27Phe, Ala70Lys, Arg72Ala and Ala77Phe were >2-fold higher than that of the wild type, indicating the substituted residues to be involved in substrate interaction. The study demonstrates that residues Phe23, Val27 and Ala70 are involved in substrate interaction, while Arg72 and Arg93 interact with other residues within the protein to provide it a functional conformation. PMID:27417238

  1. Tyrosine Residues from the S4-S5 Linker of Kv11.1 Channels Are Critical for Slow Deactivation.

    PubMed

    Ng, Chai-Ann; Gravel, Andrée E; Perry, Matthew D; Arnold, Alexandre A; Marcotte, Isabelle; Vandenberg, Jamie I

    2016-08-12

    Slow deactivation of Kv11.1 channels is critical for its function in the heart. The S4-S5 linker, which joins the voltage sensor and pore domains, plays a critical role in this slow deactivation gating. Here, we use NMR spectroscopy to identify the membrane-bound surface of the S4S5 linker, and we show that two highly conserved tyrosine residues within the KCNH subfamily of channels are membrane-associated. Site-directed mutagenesis and electrophysiological analysis indicates that Tyr-542 interacts with both the pore domain and voltage sensor residues to stabilize activated conformations of the channel, whereas Tyr-545 contributes to the slow kinetics of deactivation by primarily stabilizing the transition state between the activated and closed states. Thus, the two tyrosine residues in the Kv11.1 S4S5 linker play critical but distinct roles in the slow deactivation phenotype, which is a hallmark of Kv11.1 channels. PMID:27317659

  2. Critical tissue residue approach linking accumulated metals in aquatic insects to population and community-level effects

    USGS Publications Warehouse

    Schmidt, Travis S.; Clements, William H.; Zuellig, Robert E.; Mitchell, Katharine A.; Church, Stanley E.; Wanty, Richard B.; San Juan, Carma A.; Adams, Monique; Lamothe, Paul J.

    2011-01-01

    Whole body Zn concentrations in individuals (n = 825) from three aquatic insect taxa (mayflies Rhithrogena spp. and Drunella spp. and the caddisfly Arctopsyche grandis) were used to predict effects on populations and communities (n = 149 samples). Both mayflies accumulated significantly more Zn than the caddisfly. The presence/absence of Drunella spp. most reliably distinguished sites with low and high Zn concentrations; however, population densities of mayflies were more sensitive to increases in accumulated Zn. Critical tissue residues (634 (mu or u)g/g Zn for Drunella spp. and 267 (mu or u)g/g Zn for Rhithrogena spp.) caused a 20% reduction in maximum (90th quantile) mayfly densities. These critical tissue residues were associated with exposure to 7.0 and 3.9 (mu or u)g/L dissolved Zn for Drunella spp. and Rhithrogena spp., respectively. A threshold in a measure of taxonomic completeness (observed/expected) was observed at 5.4 (mu or u)g/L dissolved Zn. Dissolved Zn concentrations associated with critical tissue residues in mayflies were also associated with adverse effects in the aquatic community as a whole. These effects on populations and communities occurred at Zn concentrations below the U.S. EPA hardness-adjusted continuous chronic criterion.

  3. Identification of Critical Paraoxonase 1 Residues Involved in High Density Lipoprotein Interaction.

    PubMed

    Gu, Xiaodong; Huang, Ying; Levison, Bruce S; Gerstenecker, Gary; DiDonato, Anthony J; Hazen, Leah B; Lee, Joonsue; Gogonea, Valentin; DiDonato, Joseph A; Hazen, Stanley L

    2016-01-22

    Paraoxonase 1 (PON1) is a high density lipoprotein (HDL)-associated protein with atherosclerosis-protective and systemic anti-oxidant functions. We recently showed that PON1, myeloperoxidase, and HDL bind to one another in vivo forming a functional ternary complex (Huang, Y., Wu, Z., Riwanto, M., Gao, S., Levison, B. S., Gu, X., Fu, X., Wagner, M. A., Besler, C., Gerstenecker, G., Zhang, R., Li, X. M., Didonato, A. J., Gogonea, V., Tang, W. H., et al. (2013) J. Clin. Invest. 123, 3815-3828). However, specific residues on PON1 involved in the HDL-PON1 interaction remain unclear. Unambiguous identification of protein residues involved in docking interactions to lipid surfaces poses considerable methodological challenges. Here we describe a new strategy that uses a novel synthetic photoactivatable and click chemistry-taggable phospholipid probe, which, when incorporated into HDL, was used to identify amino acid residues on PON1 that directly interact with the lipoprotein phospholipid surface. Several specific PON1 residues (Leu-9, Tyr-185, and Tyr-293) were identified through covalent cross-links with the lipid probes using affinity isolation coupled to liquid chromatography with on-line tandem mass spectrometry. Based upon the crystal structure for PON1, the identified residues are all localized in relatively close proximity on the surface of PON1, defining a domain that binds to the HDL lipid surface. Site-specific mutagenesis of the identified PON1 residues (Leu-9, Tyr-185, and Tyr-293), coupled with functional studies, reveals their importance in PON1 binding to HDL and both PON1 catalytic activity and stability. Specifically, the residues identified on PON1 provide important structural insights into the PON1-HDL interaction. More generally, the new photoactivatable and affinity-tagged lipid probe developed herein should prove to be a valuable tool for identifying contact sites supporting protein interactions with lipid interfaces such as found on cell membranes

  4. Identification of residues on human receptor DPP4 critical for MERS-CoV binding and entry

    SciTech Connect

    Song, Wenfei; Wang, Ying; Wang, Nianshuang; Wang, Dongli; Guo, Jianying; Fu, Lili; Shi, Xuanling

    2014-12-15

    Middle East respiratory syndrome coronavirus (MERS-CoV) infects host cells through binding the receptor binding domain (RBD) on its spike glycoprotein to human receptor dipeptidyl peptidase 4 (hDPP4). Here, we report identification of critical residues on hDPP4 for RBD binding and virus entry through analysis of a panel of hDPP4 mutants. Based on the RBD–hDPP4 crystal structure we reported, the mutated residues were located at the interface between RBD and hDPP4, which potentially changed the polarity, hydrophobic or hydrophilic properties of hDPP4, thereby interfering or disrupting their interaction with RBD. Using surface plasmon resonance (SPR) binding analysis and pseudovirus infection assay, we showed that several residues in hDPP4–RBD binding interface were important on hDPP4–RBD binding and viral entry. These results provide atomic insights into the features of interactions between hDPP4 and MERS-CoV RBD, and also provide potential explanation for cellular and species tropism of MERS-CoV infection. - Highlights: • It has been demonstrated that MERS-CoV infects host cells through binding its envelope spike (S) glycoprotein to the host cellular receptor dipeptidyl peptidase 4 (DPP4). • To identify the critical residues on hDPP4 for RBD binding and virus entry, we constructed a panel of hDPP4 mutants based on structure-guided mutagenesis. • Using surface plasmon resonance (SPR) binding analysis and pseudovirus infection assay, we showed that several residues on hDPP4 had significant impacts on virus/receptor interactions and viral entry. • Our study has provided new insights into the features of interactions between hDPP4 and MERS-CoV RBD, and provides potential explanation for cellular and species tropism of MERS-CoV infection.

  5. Synthesis of lysine methyltransferase inhibitors

    PubMed Central

    Hui, Chunngai; Ye, Tao

    2015-01-01

    Lysine methyltransferase which catalyze methylation of histone and non-histone proteins, play a crucial role in diverse biological processes and has emerged as a promising target for the development of various human diseases, including cancer, inflammation, and psychiatric disorders. However, inhibiting lysine methyltransferases selectively has presented many challenges to medicinal chemists. During the past decade, lysine methyltransferase inhibitors covering many different structural classes have been designed and developed. In this review, we describe the development of selective, small-molecule inhibitors of lysine methyltransferases with an emphasis on their discovery and chemical synthesis. We highlight the current state of lysine methyltransferase inhibitors and discuss future directions and opportunities for lysine methyltransferase inhibitor discovery. PMID:26258118

  6. Substrate specificity and mapping of residues critical for transport in the high-affinity glutathione transporter Hgt1p.

    PubMed

    Zulkifli, Mohammad; Yadav, Shambhu; Thakur, Anil; Singla, Shiffalli; Sharma, Monika; Bachhawat, Anand Kumar

    2016-08-01

    The high-affinity glutathione transporter Hgt1p of Saccharomyces cerevisiae belongs to a relatively new and structurally uncharacterized oligopeptide transporter (OPT) family. To understand the structural features required for interaction with Hgt1p, a quantitative investigation of substrate specificity of Hgt1p was carried out. Hgt1p showed a higher affinity for reduced glutathione (GSH), whereas it transported oxidized glutathione (GSSG) and other glutathione conjugates with lower affinity. To identify the residues of Hgt1p critical for substrate binding and translocation, all amino acid residues of the 13 predicted transmembrane domains (TMDs) have been subjected to mutagenesis. Functional evaluation of these 269 mutants by growth and biochemical assay followed by kinetic analysis of the severely defective mutants including previous mutagenic studies on this transporter have led to the identification of N124 (TMD1), V185 (TMD3), Q222, G225 and Y226 (TMD4), P292 (TMD5), Y374 (TMD6), L429 (TMD7) and F523 and Q526 (TMD9) as critical for substrate binding with at least 3-fold increase in Km upon mutagenesis to alanine. In addition residues Y226 and Y374 appeared to be important for differential substrate specificity. An ab initio model of Hgt1p was built and refined using these mutagenic data that yielded a helical arrangement that includes TMD3, TMD4, TMD5, TMD6, TMD7, TMD9 and TMD13 as pore-lining helices with the functionally important residues in a channel-facing orientation. Taken together the results of this study provides the first mechanistic insights into glutathione transport by a eukaryotic high-affinity glutathione transporter. PMID:27252386

  7. A conserved amino acid residue critical for product and substrate specificity in plant triterpene synthases.

    PubMed

    Salmon, Melissa; Thimmappa, Ramesha B; Minto, Robert E; Melton, Rachel E; Hughes, Richard K; O'Maille, Paul E; Hemmings, Andrew M; Osbourn, Anne

    2016-07-26

    Triterpenes are structurally complex plant natural products with numerous medicinal applications. They are synthesized through an origami-like process that involves cyclization of the linear 30 carbon precursor 2,3-oxidosqualene into different triterpene scaffolds. Here, through a forward genetic screen in planta, we identify a conserved amino acid residue that determines product specificity in triterpene synthases from diverse plant species. Mutation of this residue results in a major change in triterpene cyclization, with production of tetracyclic rather than pentacyclic products. The mutated enzymes also use the more highly oxygenated substrate dioxidosqualene in preference to 2,3-oxidosqualene when expressed in yeast. Our discoveries provide new insights into triterpene cyclization, revealing hidden functional diversity within triterpene synthases. They further open up opportunities to engineer novel oxygenated triterpene scaffolds by manipulating the precursor supply. PMID:27412861

  8. A conserved amino acid residue critical for product and substrate specificity in plant triterpene synthases

    PubMed Central

    Salmon, Melissa; Thimmappa, Ramesha B.; Minto, Robert E.; Melton, Rachel E.; O’Maille, Paul E.; Hemmings, Andrew M.; Osbourn, Anne

    2016-01-01

    Triterpenes are structurally complex plant natural products with numerous medicinal applications. They are synthesized through an origami-like process that involves cyclization of the linear 30 carbon precursor 2,3-oxidosqualene into different triterpene scaffolds. Here, through a forward genetic screen in planta, we identify a conserved amino acid residue that determines product specificity in triterpene synthases from diverse plant species. Mutation of this residue results in a major change in triterpene cyclization, with production of tetracyclic rather than pentacyclic products. The mutated enzymes also use the more highly oxygenated substrate dioxidosqualene in preference to 2,3-oxidosqualene when expressed in yeast. Our discoveries provide new insights into triterpene cyclization, revealing hidden functional diversity within triterpene synthases. They further open up opportunities to engineer novel oxygenated triterpene scaffolds by manipulating the precursor supply. PMID:27412861

  9. Multiple lysine methylation of PCAF by Set9 methyltransferase

    SciTech Connect

    Masatsugu, Toshihiro; Yamamoto, Ken

    2009-03-27

    The molecular functions of several non-histone proteins are regulated through lysine modification by histone methyltransferases. The p300/CBP-associated factor (PCAF) is an acetyltransferase that has been implicated in many cellular processes. Here, we report that PCAF is a novel substrate of Set9 methyltransferase. In vitro mapping experiments revealed six lysine residues could be methylated by Set9. A comparison of amino acid sequences of target sites revealed the novel consensus motif which differs from previously identified Set9-consensus sequence. Further methyltransferase assays focusing on the six lysine residues showed that K78 and K89 are preferentially methylated in full-length PCAF in vitro. Using specific antibodies recognizing mono-methylated K89, in vivo PCAF methylation and its nuclear localization were demonstrated. Our data may lead to a new insight into PCAF functions and provide additional information to identify unknown targets of Set9.

  10. N-terminal basic amino acid residues of Beet black scorch virus capsid protein play a critical role in virion assembly and systemic movement

    PubMed Central

    2013-01-01

    Background Beet black scorch virus (BBSV) is a small single-stranded, positive-sense RNA plant virus belonging to the genus Necrovirus, family Tombusviridae. Its capsid protein (CP) contains a 13 amino acid long basic region at the N-terminus, rich in arginine and lysine residues, which is thought to interact with viral RNA to initiate virion assembly. Results In the current study, a series of BBSV mutants containing amino acid substitutions as well as deletions within the N-terminal region were generated and examined for their effects on viral RNA replication, virion assembly, and long distance spread in protoplasts and whole host plants of BBSV. The RNA-binding activities of the mutated CPs were also evaluated in vitro. These experiments allowed us to identify two key basic amino acid residues in this region that are responsible for initiating virus assembly through RNA-binding. Proper assembly of BBSV particles is in turn needed for efficient viral systemic movement. Conclusions We have identified two basic amino acid residues near the N-terminus of the BBSV CP that bind viral RNA with high affinity to initiate virion assembly. We further provide evidence showing that systemic spread of BBSV in infected plants requires intact virions. This study represents the first in-depth investigation of the role of basic amino acid residues within the N-terminus of a necroviral CP. PMID:23786675

  11. Characterization and crystal structure of lysine insensitive Corynebacterium glutamicum dihydrodipicolinate synthase (cDHDPS) protein

    SciTech Connect

    Rice, E.A.; Bannon, G.A.; Glenn, K.C.; Jeong, S.S.; Sturman, E.J.; Rydel, T.J.

    2008-11-21

    The lysine insensitive Corynebacterium glutamicum dihydrodipicolinate synthase enzyme (cDHDPS) was recently successfully introduced into maize plants to enhance the level of lysine in the grain. To better understand lysine insensitivity of the cDHDPS, we expressed, purified, kinetically characterized the protein, and solved its X-ray crystal structure. The cDHDPS enzyme has a fold and overall structure that is highly similar to other DHDPS proteins. A noteworthy feature of the active site is the evidence that the catalytic lysine residue forms a Schiff base adduct with pyruvate. Analyses of the cDHDPS structure in the vicinity of the putative binding site for S-lysine revealed that the allosteric binding site in the Escherichia coli DHDPS protein does not exist in cDHDPS due to three non-conservative amino acids substitutions, and this is likely why cDHDPS is not feedback inhibited by lysine.

  12. Homology modeling, substrate docking, and molecular simulation studies of mycobacteriophage Che12 lysin A.

    PubMed

    Saadhali, Shainaba A; Hassan, Sameer; Hanna, Luke Elizabeth; Ranganathan, Uma Devi; Kumar, Vanaja

    2016-08-01

    Mycobacteriophages produce lysins that break down the host cell wall at the end of lytic cycle to release their progenies. The ability to lyse mycobacterial cells makes the lysins significant. Mycobacteriophage Che12 is the first reported temperate phage capable of infecting and lysogenising Mycobacterium tuberculosis. Gp11 of Che12 was found to have Chitinase domain that serves as endolysin (lysin A) for Che12. Structure of gp11 was modeled and evaluated using Ramachandran plot in which 98 % of the residues are in the favored and allowed regions. Che12 lysin A was predicted to act on NAG-NAM-NAG molecules in the peptidoglycan of cell wall. The tautomers of NAG-NAM-NAG molecule were generated and docked with lysin A. The stability and binding affinity of lysin A - NAG-NAM-NAG tautomers were studied using molecular dynamics simulations. PMID:27411553

  13. Critical body residue of compounds having different mode of action on energy metabolism in benthic invertebrates

    SciTech Connect

    Penttinen, O.P.; Kukkonen, J.

    1995-12-31

    The toxicity of organic chemicals with different mode of toxic action was evaluated by determining their effect on the metabolic rate of two common benthic invertebrates, midge larva (Chironomus riparius) and oligochate worm (Lumbriculus variegatus). The rate of metabolism was monitored by direct microcalorimetry and the change of heat output was related to the body residue of chemicals. The expected response of 2,4,5-trichlorophenol (TCP), known as an uncoupler of oxidative phosphorylation, was an increase of metabolic rate. The animals were exposed 24 h to water spiked with TCP (10 to 1,200 {micro}g/L) and they received the body residues of TCP in the range of 8.8 to 336 {micro}g/g wet wt (0.04 to 1.75 {micro}mol/g). The threshold concentration was 0.7 {micro}mol/g wet wt. (C. riparius) or 1.0 {micro}mol/g wet wt. (L. variegatus) above which the rate of heat dissipation increased in direct proportion to the concentration of TCP in tissue. At maximum, the metabolic rate increased by a factor of three. At the highest water concentration animals were dying and the metabolic rate was low. The energetic responses obtained with TCP are compared to those of a non-polar narcotic compound 1,2,4-trichlorobenzene and an other uncoupling agent, 2,4-dinitrophenol.

  14. Tropomyosin lysine reactivities and relationship to coiled-coil structure.

    PubMed

    Hitchcock-DeGregori, S E; Lewis, S F; Chou, T M

    1985-06-18

    We have carried out a detailed analysis of tropomyosin structure using lysines as specific probes for the protein surface in regions of the molecule that have not been investigated by other methods. We have measured the relative reactivities of lysines in rabbit skeletal muscle alpha, alpha-tropomyosin with acetic anhydride using a competitive labeling procedure. We have identified 37 of 39 lysines and find that they range 20-fold in reactivity. The observed reactivities are related to the coiled-coil model of the tropomyosin molecule [Crick, F.H.C. (1953) Acta Crystallogr. 6, 689-697; McLachlan, A.D., Stewart, M., & Smillie, L.B. (1975) J. Mol. Biol. 98, 281-291] and other available chemical and physical information about the structure. In most cases, the observed lysine reactivities can be explained by allowable interactions with neighboring amino acid side chains on the same or facing alpha-helix. However, we found no correlation between reactivity and helical position of a given lysine. For example, lysines in the outer helical positions included lysines of low as well as high reactivity, indicating that they vary widely in their accessibility to solvent and that the coiled coil is heterogeneous along its length. Furthermore, the middle of the molecule (residues 126-182) that is susceptible to proteolysis and known to be the least stable region of the protein also contains some of the least and most reactive lysines. We have discussed the implications of our results on our understanding the structures of tropomyosin and other coiled-coil proteins as well as globular proteins containing helical regions. PMID:3927977

  15. Mutational analysis of GlnB residues critical for NifA activation in Azospirillum brasilense.

    PubMed

    Inaba, Juliana; Thornton, Jeremy; Huergo, Luciano Fernandes; Monteiro, Rose Adele; Klassen, Giseli; Pedrosa, Fábio de Oliveira; Merrick, Mike; de Souza, Emanuel Maltempi

    2015-02-01

    PII proteins are signal transduction that sense cellular nitrogen status and relay this signals to other targets. Azospirillum brasilense is a nitrogen fixing bacterium, which associates with grasses and cereals promoting beneficial effects on plant growth and crop yields. A. brasilense contains two PII encoding genes, named glnB and glnZ. In this paper, glnB was mutagenised in order to identify amino acid residues involved in GlnB signaling. Two variants were obtained by random mutagenesis, GlnBL13P and GlnBV100A and a site directed mutant, GlnBY51F, was obtained. Their ability to complement nitrogenase activity of glnB mutant strains of A. brasilense were determined. The variant proteins were also overexpressed in Escherichia coli, purified and characterized biochemically. None of the GlnB variant forms was able to restore nitrogenase activity in glnB mutant strains of A. brasilense LFH3 and 7628. The purified GlnBY51F and GlnBL13P proteins could not be uridylylated by GlnD, whereas GlnBV100A was uridylylated but at only 20% of the rate for wild type GlnB. Biochemical and computational analyses suggest that residue Leu13, located in the α helix 1 of GlnB, is important to maintain GlnB trimeric structure and function. The substitution V100A led to a lower affinity for ATP binding. Together the results suggest that NifA activation requires uridylylated GlnB bound to ATP. PMID:25644954

  16. Pilin and Sortase Residues Critical for Endocarditis- and Biofilm-Associated Pilus Biogenesis in Enterococcus faecalis

    PubMed Central

    Nielsen, Hailyn V.; Flores-Mireles, Ana L.; Kau, Andrew L.; Kline, Kimberly A.; Pinkner, Jerome S.; Neiers, Fabrice; Normark, Staffan; Henriques-Normark, Birgitta

    2013-01-01

    Enterococci commonly cause hospital-acquired infections, such as infective endocarditis and catheter-associated urinary tract infections. In animal models of these infections, a long hairlike extracellular protein fiber known as the endocarditis- and biofilm-associated (Ebp) pilus is an important virulence factor for Enterococcus faecalis. For Ebp and other sortase-assembled pili, the pilus-associated sortases are essential for fiber formation as they create covalent isopeptide bonds between the sortase recognition motif and the pilin-like motif of the pilus subunits. However, the molecular requirements governing the incorporation of the three pilus subunits (EbpA, EbpB, and EbpC) have not been investigated in E. faecalis. Here, we show that a Lys residue within the pilin-like motif of the EbpC subunit was necessary for EbpC polymerization. However, incorporation of EbpA into the pilus fiber only required its sortase recognition motif (LPXTG), while incorporation of EbpB only required its pilin-like motif. Only the sortase recognition motif would be required for incorporation of the pilus tip subunit, while incorporation of the base subunit would only require the pilin recognition motif. Thus, these data support a model with EbpA at the tip and EbpB at the base of an EbpC polymer. In addition, the housekeeping sortase, SrtA, was found to process EbpB and its predicted catalytic Cys residue was required for efficient cell wall anchoring of mature Ebp pili. Thus, we have defined molecular interactions involved in fiber polymerization, minor subunit organization, and pilus subcellular compartmentalization in the E. faecalis Ebp pilus system. These studies advance our understanding of unique molecular mechanisms of sortase-assembled pilus biogenesis. PMID:23913319

  17. Comprehensive profiling of lysine acetylproteome analysis reveals diverse functions of lysine acetylation in common wheat.

    PubMed

    Zhang, Yumei; Song, Limin; Liang, Wenxing; Mu, Ping; Wang, Shu; Lin, Qi

    2016-01-01

    Lysine acetylation of proteins, a dynamic and reversible post-translational modification, plays a critical regulatory role in both eukaryotes and prokaryotes. Several researches have been carried out on acetylproteome in plants. However, until now, there have been no data on common wheat, the major cereal crop in the world. In this study, we performed a global acetylproteome analysis of common wheat variety (Triticum aestivum L.), Chinese Spring. In total, 416 lysine modification sites were identified on 277 proteins, which are involved in a wide variety of biological processes. Consistent with previous studies, a large proportion of the acetylated proteins are involved in metabolic process. Interestingly, according to the functional enrichment analysis, 26 acetylated proteins are involved in photosynthesis and Calvin cycle, suggesting an important role of lysine acetylation in these processes. Moreover, protein interaction network analysis reveals that diverse interactions are modulated by protein acetylation. These data represent the first report of acetylome in common wheat and serve as an important resource for exploring the physiological role of lysine acetylation in this organism and likely in all plants. PMID:26875666

  18. Comprehensive profiling of lysine acetylproteome analysis reveals diverse functions of lysine acetylation in common wheat

    PubMed Central

    Zhang, Yumei; Song, Limin; Liang, Wenxing; Mu, Ping; Wang, Shu; Lin, Qi

    2016-01-01

    Lysine acetylation of proteins, a dynamic and reversible post-translational modification, plays a critical regulatory role in both eukaryotes and prokaryotes. Several researches have been carried out on acetylproteome in plants. However, until now, there have been no data on common wheat, the major cereal crop in the world. In this study, we performed a global acetylproteome analysis of common wheat variety (Triticum aestivum L.), Chinese Spring. In total, 416 lysine modification sites were identified on 277 proteins, which are involved in a wide variety of biological processes. Consistent with previous studies, a large proportion of the acetylated proteins are involved in metabolic process. Interestingly, according to the functional enrichment analysis, 26 acetylated proteins are involved in photosynthesis and Calvin cycle, suggesting an important role of lysine acetylation in these processes. Moreover, protein interaction network analysis reveals that diverse interactions are modulated by protein acetylation. These data represent the first report of acetylome in common wheat and serve as an important resource for exploring the physiological role of lysine acetylation in this organism and likely in all plants. PMID:26875666

  19. Critical Assessment of the Important Residues Involved in the Dimerization and Catalysis of MERS Coronavirus Main Protease

    PubMed Central

    Ho, Bo-Lin; Cheng, Shu-Chun; Shi, Lin; Wang, Ting-Yun; Ho, Kuan-I; Chou, Chi-Yuan

    2015-01-01

    Background A highly pathogenic human coronavirus (CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), has emerged in Jeddah and other places in Saudi Arabia, and has quickly spread to European and Asian countries since September 2012. Up to the 1st October 2015 it has infected at least 1593 people with a global fatality rate of about 35%. Studies to understand the virus are necessary and urgent. In the present study, MERS-CoV main protease (Mpro) is expressed; the dimerization of the protein and its relationship to catalysis are investigated. Methods and Results The crystal structure of MERS-CoV Mpro indicates that it shares a similar scaffold to that of other coronaviral Mpro and consists of chymotrypsin-like domains I and II and a helical domain III of five helices. Analytical ultracentrifugation analysis demonstrated that MERS-CoV Mpro undergoes a monomer to dimer conversion in the presence of a peptide substrate. Glu169 is a key residue and plays a dual role in both dimerization and catalysis. The mutagenesis of other residues found on the dimerization interface indicate that dimerization of MERS-CoV Mpro is required for its catalytic activity. One mutation, M298R, resulted in a stable dimer with a higher level of proteolytic activity than the wild-type enzyme. Conclusions MERS-CoV Mpro shows substrate-induced dimerization and potent proteolytic activity. A critical assessment of the residues important to these processes provides insights into the correlation between dimerization and catalysis within the coronaviral Mpro family. PMID:26658006

  20. Identification by mutational analysis of four critical residues in the molybdenum cofactor domain of eukaryotic nitrate reductase.

    PubMed

    Meyer, C; Gonneau, M; Caboche, M; Rouzé, P

    1995-08-21

    The nucleotide sequence of the nitrate reductase (NR) molybdenum cofactor (MoCo) domain was determined in four Nicotiana plumbaginifolia mutants affected in the NR apoenzyme gene. In each case, missense mutations were found in the MoCo domain which affected amino acids that were conserved not only among eukaryotic NRs but also in animal sulfite oxidase sequences. Moreover an abnormal NR molecular mass was observed in three mutants, suggesting that the integrity of the MoCo domain is essential for a proper assembly of holo-NR. These data allowed to pinpoint critical residues in the NR MoCo domain necessary for the enzyme activity but also important for its quaternary structure. PMID:7656976

  1. Mapping and genotypic analysis of NK-lysin gene in chicken

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Antimicrobial peptides (AMP) are important elements of the first line of defence against pathogens in animals. NK-lysin is a cationic AMP that plays a critical role in innate immunity. The chicken NK-lysin gene has been cloned and its antimicrobial and anticancer activity has been descri...

  2. Mapping and genotypic analysis of NK-lysin gene in chicken

    Technology Transfer Automated Retrieval System (TEKTRAN)

    NK-lysin is a cationic anti-microbial peptide that plays a critical role in innate immunity against infectious pathogens. Chicken NK-lysin has been cloned and its antimicrobial and anticancer activity has been described but its location in the chicken genome prior this study was unknown. A 6000 rad ...

  3. Characterization of the fibrinogen binding domain of bacteriophage lysin from Streptococcus mitis.

    PubMed

    Seo, Ho Seong; Sullam, Paul M

    2011-09-01

    The binding of bacteria to human platelets is a likely central mechanism in the pathogenesis of infective endocarditis. Platelet binding by Streptococcus mitis SF100 is mediated in part by a lysin encoded by the lysogenic bacteriophage SM1. In addition to its role in the phage life cycle, lysin mediates the binding of S. mitis to human platelets via its interaction with fibrinogen on the platelet surface. To better define the region of lysin mediating fibrinogen binding, we tested a series of purified lysin truncation variants for their abilities to bind this protein. These studies revealed that the fibrinogen binding domain of lysin is contained within the region spanned by amino acid residues 102 to 198 (lysin(102-198)). This region has no sequence homology to other known fibrinogen binding proteins. Lysin(102-198) bound fibrinogen comparably to full-length lysin and with the same selectivity for the fibrinogen Aα and Bβ chains. Lysin(102-198) also inhibited the binding in vitro of S. mitis to human fibrinogen and platelets. When assessed by platelet aggregometry, the disruption of the lysin gene in SF100 resulted in a significantly longer time to the onset of aggregation of human platelets than that of the parent strain. The preincubation of platelets with purified lysin(102-198) also delayed the onset of aggregation by SF100. These results indicate that the binding of lysin to fibrinogen is mediated by a specific domain of the phage protein and that this interaction is important for both platelet binding and aggregation by S. mitis. PMID:21690235

  4. Mechanism of Lysine 48 Selectivity during Polyubiquitin Chain Formation by the Ube2R1/2 Ubiquitin-Conjugating Enzyme.

    PubMed

    Hill, Spencer; Harrison, Joseph S; Lewis, Steven M; Kuhlman, Brian; Kleiger, Gary

    2016-06-01

    Lysine selectivity is of critical importance during polyubiquitin chain formation because the identity of the lysine controls the biological outcome. Ubiquitins are covalently linked in polyubiquitin chains through one of seven lysine residues on its surface and the C terminus of adjacent protomers. Lys 48-linked polyubiquitin chains signal for protein degradation; however, the structural basis for Lys 48 selectivity remains largely unknown. The ubiquitin-conjugating enzyme Ube2R1/2 has exquisite specificity for Lys 48, and computational docking of Ube2R1/2 and ubiquitin predicts that Lys 48 is guided to the active site through a key electrostatic interaction between Arg 54 on ubiquitin and Asp 143 on Ube2R1/2. The validity of this interaction was confirmed through biochemical experiments. Since structural examples involving Arg 54 in protein-ubiquitin complexes are exceedingly rare, these results provide additional insight into how ubiquitin-protein complexes can be stabilized. We discuss how these findings relate to how other ubiquitin-conjugating enzymes direct the lysine specificity of polyubiquitin chains. PMID:27044868

  5. The HIV-1 Tat Protein Is Monomethylated at Lysine 71 by the Lysine Methyltransferase KMT7.

    PubMed

    Ali, Ibraheem; Ramage, Holly; Boehm, Daniela; Dirk, Lynnette M A; Sakane, Naoki; Hanada, Kazuki; Pagans, Sara; Kaehlcke, Katrin; Aull, Katherine; Weinberger, Leor; Trievel, Raymond; Schnoelzer, Martina; Kamada, Masafumi; Houtz, Robert; Ott, Melanie

    2016-07-29

    The HIV-1 transactivator protein Tat is a critical regulator of HIV transcription primarily enabling efficient elongation of viral transcripts. Its interactions with RNA and various host factors are regulated by ordered, transient post-translational modifications. Here, we report a novel Tat modification, monomethylation at lysine 71 (K71). We found that Lys-71 monomethylation (K71me) is catalyzed by KMT7, a methyltransferase that also targets lysine 51 (K51) in Tat. Using mass spectrometry, in vitro enzymology, and modification-specific antibodies, we found that KMT7 monomethylates both Lys-71 and Lys-51 in Tat. K71me is important for full Tat transactivation, as KMT7 knockdown impaired the transcriptional activity of wild type (WT) Tat but not a Tat K71R mutant. These findings underscore the role of KMT7 as an important monomethyltransferase regulating HIV transcription through Tat. PMID:27235396

  6. PlyC: A multimeric bacteriophage lysin

    PubMed Central

    Nelson, Daniel; Schuch, Raymond; Chahales, Peter; Zhu, Shiwei; Fischetti, Vincent A.

    2006-01-01

    Lysins are murein hydrolases produced by bacteriophage that act on the bacterial host cell wall to release progeny phage. When added extrinsically in their purified form, these enzymes produce total lysis of susceptible Gram-positive bacteria within seconds, suggesting a unique antimicrobial strategy. All known Gram-positive lysins are produced as a single polypeptide containing a catalytic activity domain, which cleaves one of the four major peptidoglycan bonds, and a cell-wall-binding domain, which may bind a species-specific carbohydrate epitope in the cell wall. Here, we have cloned and expressed a unique lysin from the streptococcal bacteriophage C1, termed PlyC. Molecular characterization of the plyC operon reveals that PlyC is, surprisingly, composed of two separate gene products, PlyCA and PlyCB. Based on biochemical and biophysical studies, the catalytically active PlyC holoenzyme is composed of eight PlyCB subunits for each PlyCA. Inhibitor studies predicted the presence of an active-site cysteine, and bioinformatic analysis revealed a cysteine, histidine-dependent amidohydrolase/peptidase domain within PlyCA. Point mutagenesis confirmed that PlyCA is responsible for the observed catalytic activity, and Cys-333 and His-420 are the active-site residues. PlyCB was found to self-assemble into an octamer, and this complex alone was able to direct streptococcal cell-wall-specific binding. Similar to no other proteins in sequence databases, PlyC defines a previously uncharacterized structural family of cell-wall hydrolases. PMID:16818874

  7. Histone H4 Lysine 20 (H4K20) Methylation, Expanding the Signaling Potential of the Proteome One Methyl Moiety at a Time.

    PubMed

    van Nuland, Rick; Gozani, Or

    2016-03-01

    Covalent post-translational modifications (PTMs) of proteins can regulate the structural and functional state of a protein in the absence of primary changes in the underlying sequence. Common PTMs include phosphorylation, acetylation, and methylation. Histone proteins are critical regulators of the genome and are subject to a highly abundant and diverse array of PTMs. To highlight the functional complexity added to the proteome by lysine methylation signaling, here we will focus on lysine methylation of histone proteins, an important modification in the regulation of chromatin and epigenetic processes. We review the signaling pathways and functions associated with a single residue, H4K20, as a model chromatin and clinically important mark that regulates biological processes ranging from the DNA damage response and DNA replication to gene expression and silencing. PMID:26598646

  8. Critical body residues for pentachlorophenol in the zebra mussel under varying conditions of pH and temperature

    SciTech Connect

    Fisher, S.W.; Hwang, H.; Atanasoff, M.; Landrum, P.F.

    1995-12-31

    The toxicity of pentachlorophenol (PCP), an ionizable phenol, is strongly dependent on environmental pH and temperature. Using the invertebrate species, the zebra mussel (Dreissena polymorpha), the authors tested whether CBRs could be used to resolve the differences in toxicity under varying conditions. The authors simultaneously measured acute toxicity and tissue concentrations of PCP under 9 different combinations of pH and temperature. CBRs were determined from tissue residues as LD{sub 50}, values and were also calculated from LC{sub 50}s in conjunction with toxicokinetic parameters determined under each set of conditions. The data show that when toxicity is based on aqueous concentrations of PCP needed to cause mortality (LC{sub 50}s), that the resulting LC{sub 50}s varied by a factor of 372 X across the range of conditions tested. However, when LD{sub 50}s were calculated from tissue residues in the mussel, the latter varied only by a factor of 12.7 across the range of conditions examined. When CBRs were determined toxicokinetically, instead of from direct measurement of tissue concentrations, these CBRs were both higher than the measured LD{sub 50}s and more variable. The authors believe this is a result of the animals having a higher filtering rate and a greater tolerance for PCP in the short-term exposures from which the toxicokinetic values were obtained. Their data generally support the utility of CBRs in minimizing variation attributable to environmental variables but also demonstrate that the method of determining a CBR will be critical.

  9. Evaluation of critical current density and residual resistance ratio limits in powder in tube Nb3Sn conductors

    NASA Astrophysics Data System (ADS)

    Segal, Christopher; Tarantini, Chiara; Hawn Sung, Zu; Lee, Peter J.; Sailer, Bernd; Thoener, Manfred; Schlenga, Klaus; Ballarino, Amalia; Bottura, Luca; Bordini, Bernardo; Scheuerlein, Christian; Larbalestier, David C.

    2016-08-01

    High critical current density (J c) Nb3Sn A15 multifilamentary wires require a large volume fraction of small grain (SG), superconducting A15 phase, as well as Cu stabilizer with high Residual Resistance Ratio (RRR) to provide electromagnetic stabilization and protection. In powder-in-tube (PIT) wires the unreacted Nb7.5 wt%Ta outer layer of the tubular filaments acts as a diffusion barrier and protects the interfilamentary Cu stabilizer from Sn contamination. A high RRR requirement generally imposes a restricted A15 reaction heat treatment to prevent localized full reaction of the filament that could allow Sn to reach the Cu. In this study we investigate recent high quality PIT wires that achieve a J c (12 T, 4.2 K) up to ∼2500 A mm‑2 and find that the minimum diffusion barrier thickness decreases as the filament aspect ratio increases from ∼1 in the inner rings of filaments to 1.3 in the outer filament rings. We found that just 2–3 diffusion barrier breaches can degrade RRR from 300 to 150 or less. Using progressive etching of the Cu we also found that the RRR degradation is localized near the external filaments where deformation is highest. Consequently minimizing filament distortion during strand fabrication is important for reducing RRR degradation. The additional challenge of developing the highest possible J c must be addressed by forming the maximum fraction of high J c SG A15 and minimizing low J c large-grain (LG) A15 morphologies. In one wire we found that 15% of the filaments had a significantly enhanced SG/LG A15 ratio and no residual A15 in the core, a feature that opens a path to substantial J c improvement.

  10. Probing the chemical mechanism and critical regulatory amino acid residues of Drosophila melanogaster arylalkylamine N-acyltransferase like 2.

    PubMed

    Dempsey, Daniel R; Carpenter, Anne-Marie; Ospina, Santiago Rodriguez; Merkler, David J

    2015-11-01

    Arylalkylamine N-acyltransferase like 2 (AANATL2) catalyzes the formation of N-acylarylalkylamides from the corresponding acyl-CoA and arylalkylamine. The N-acylation of biogenic amines in Drosophila melanogaster is a critical step for the inactivation of neurotransmitters, cuticle sclerotization, and melatonin biosynthesis. In addition, D. melanogaster has been used as a model system to evaluate the biosynthesis of fatty acid amides: a family of potent cell signaling lipids. We have previously showed that AANATL2 catalyzes the formation of N-acylarylakylamides, including long-chain N-acylserotonins and N-acyldopamines. Herein, we define the kinetic mechanism for AANATL2 as an ordered sequential mechanism with acetyl-CoA binding first followed by tyramine to generate the ternary complex prior to catalysis. Bell shaped kcat,app - acetyl-CoA and (kcat/Km)app - acetyl-CoA pH-rate profiles identified two apparent pKa,app values of ∼7.4 and ∼8.9 that are critical to catalysis, suggesting the AANATL2-catalyzed formation of N-acetyltyramine occurs through an acid/base chemical mechanism. Site-directed mutagenesis of a conserved glutamate that corresponds to the catalytic base for other D. melanogaster AANATL enzymes did not produce a substantial depression in the kcat,app value nor did it abolish the pKa,app value attributed to the general base in catalysis (pKa ∼7.4). These data suggest that AANATL2 catalyzes the formation of N-acylarylalkylamides using either different catalytic residues or a different chemical mechanism relative to other D. melanogaster AANATL enzymes. In addition, we constructed other site-directed mutants of AANATL2 to help define the role of targeted amino acids in substrate binding and/or enzyme catalysis. PMID:26476413

  11. Structural Basis for l-Lysine Feedback Inhibition of Homocitrate Synthase

    SciTech Connect

    Bulfer, Stacie L.; Scott, Erin M.; Pillus, Lorraine; Trievel, Raymond C.

    2010-09-02

    The {alpha}-aminoadipate pathway of lysine biosynthesis is modulated at the transcriptional and biochemical levels by feedback inhibition. The first enzyme in the {alpha}-aminoadipate pathway, homocitrate synthase (HCS), is the target of the feedback regulation and is strongly inhibited by L-lysine. Here we report the structure of Schizosaccharomyces pombe HCS (SpHCS) in complex with L-lysine. The structure illustrates that the amino acid directly competes with the substrate 2-oxoglutarate for binding within the active site of HCS. Differential recognition of the substrate and inhibitor is achieved via a switch position within the ({alpha}/{beta}){sub 8} TIM barrel of the enzyme that can distinguish between the C5-carboxylate group of 2-oxoglutarate and the {epsilon}-ammonium group of L-lysine. In vitro and in vivo assays demonstrate that mutations of the switch residues, which interact with the L-lysine {epsilon}-ammonium group, abrogate feedback inhibition, as do substitutions of residues within the C-terminal domain that were identified in a previous study of L-lysine-insensitive HCS mutants in Saccharomyces cerevisiae. Together, these results yield new insights into the mechanism of feedback regulation of an enzyme central to lysine biosynthesis.

  12. Re-analysis of narcotic critical body residue data using the equilibrium distribution concept and refined partition coefficients.

    PubMed

    Endo, Satoshi

    2016-08-10

    Narcosis occurs as a result of the accumulation of chemicals in the phospholipid membrane. The toxic threshold concentration in the membrane is thought to be relatively constant across different chemicals and species. Hence, estimating chemical concentrations in the membrane is expected to reduce the variability of narcotic critical body residue (CBR) data. In this study, a high quality CBR dataset for three aquatic species reported recently in the literature was evaluated with the internal equilibrium distribution concept. The raw wet-weight-based CBR values were converted to membrane-weight-based CBR values by assuming that the chemical is distributed in storage lipids, membranes, proteins, and water according to the respective equilibrium partition coefficients. Several sets of partition coefficients were compared for this analysis. The results were consistent with the notion that the use of a structural protein instead of serum albumin as a surrogate for the body protein fraction could reduce the variability of CBRs. Partition coefficients predicted by polyparameter linear free energy relationships (PP-LFERs) reduced the variability of CBRs as much as or even more than experimental partition coefficients did. It is suggested that CBR data for chemicals with larger structural diversity and biological species with more distinct compositions are needed to evaluate further the equilibrium distribution concept and the constant membrane threshold hypothesis. PMID:27136717

  13. Regulation of Transcription Factor Yin Yang 1 by SET7/9-mediated Lysine Methylation

    PubMed Central

    Zhang, Wen-juan; Wu, Xiao-nan; Shi, Tao-tao; Xu, Huan-teng; Yi, Jia; Shen, Hai-feng; Huang, Ming-feng; Shu, Xing-yi; Wang, Fei-fei; Peng, Bing-ling; Xiao, Rong-quan; Gao, Wei-wei; Ding, Jian-cheng; Liu, Wen

    2016-01-01

    Yin Yang 1 (YY1) is a multifunctional transcription factor shown to be critical in a variety of biological processes. Although it is regulated by multiple types of post-translational modifications (PTMs), whether YY1 is methylated, which enzyme methylates YY1, and hence the functional significance of YY1 methylation remains completely unknown. Here we reported the first methyltransferase, SET7/9 (KMT7), capable of methylating YY1 at two highly conserved lysine (K) residues, K173 and K411, located in two distinct domains, one in the central glycine-rich region and the other in the very carboxyl-terminus. Functional studies revealed that SET7/9-mediated YY1 methylation regulated YY1 DNA-binding activity both in vitro and at specific genomic loci in cultured cells. Consistently, SET7/9-mediated YY1 methylation was shown to involve in YY1-regulated gene transcription and cell proliferation. Our findings revealed a novel regulatory strategy, methylation by lysine methyltransferase, imposed on YY1 protein, and linked YY1 methylation with its biological functions. PMID:26902152

  14. Mutational analysis of nucleoside diphosphate kinase from Pseudomonas aeruginosa: characterization of critical amino acid residues involved in exopolysaccharide alginate synthesis.

    PubMed Central

    Sundin, G W; Shankar, S; Chakrabarty, A M

    1996-01-01

    We report the utilization of site-directed and random mutagenesis procedures in the gene encoding nucleoside diphosphate kinase (ndk) from Pseudomonas aeruginosa in order to examine the role of Ndk in the production of alginate by this organism. Cellular levels of the 16-kDa form of the Ndk enzyme are greatly reduced in P. aeruginosa 8830 with a knockout mutation in the algR2 gene (8830R2::Cm); this strain is also defective in the production of the exopolysaccharide alginate. In this study, we isolated four mutations in ndk (Ala-14-->Pro [Ala14Pro], Gly21Val, His117Gln, and Ala125Arg) which resulted in the loss of Ndk biochemical activity; hyperexpression of any of these four mutant genes did not restore alginate production to 8830R2::Cm. We identified six additional amino acid residues (Ser-43, Ala-56, Ser-69, Glu-80, Gly-91, and Asp-135) whose alteration resulted in the inability of Ndk to complement alginate production. After hyperproduction in 8830R2::Cm, it was determined that each of these six mutant Ndks was biochemically active. However, in four cases, the in vivo levels of Ndk were reduced, which consequently affected the growth of 8830R2::Cm in the presence of Tween 20. Two mutant Ndk proteins which could not complement the alginate synthesis defect in 8830R2::Cm were not affected in any characteristic examined in the present study. All of the mutant Ndks characterized which were still biochemically active formed membrane complexes with Pk, resulting in GTP synthesis. Two of the four Ndk activity mutants (His117Gln and Ala125Arg) identified were capable of being truncated to 12 kDa and formed a membrane complex with Pk; however, the complexes formed were inactive for GTP synthesis. The other two Ndk activity mutants could be truncated to 12 kDa but were not detected in membrane fractions. These results further our understanding of the role of Ndk in alginate synthesis and identify amino acid residues in Ndk which have not previously been studied as

  15. Cyclophilin-B Modulates Collagen Cross-linking by Differentially Affecting Lysine Hydroxylation in the Helical and Telopeptidyl Domains of Tendon Type I Collagen.

    PubMed

    Terajima, Masahiko; Taga, Yuki; Chen, Yulong; Cabral, Wayne A; Hou-Fu, Guo; Srisawasdi, Sirivimol; Nagasawa, Masako; Sumida, Noriko; Hattori, Shunji; Kurie, Jonathan M; Marini, Joan C; Yamauchi, Mitsuo

    2016-04-29

    Covalent intermolecular cross-linking provides collagen fibrils with stability. The cross-linking chemistry is tissue-specific and determined primarily by the state of lysine hydroxylation at specific sites. A recent study on cyclophilin B (CypB) null mice, a model of recessive osteogenesis imperfecta, demonstrated that lysine hydroxylation at the helical cross-linking site of bone type I collagen was diminished in these animals (Cabral, W. A., Perdivara, I., Weis, M., Terajima, M., Blissett, A. R., Chang, W., Perosky, J. E., Makareeva, E. N., Mertz, E. L., Leikin, S., Tomer, K. B., Kozloff, K. M., Eyre, D. R., Yamauchi, M., and Marini, J. C. (2014) PLoS Genet 10, e1004465). However, the extent of decrease appears to be tissue- and molecular site-specific, the mechanism of which is unknown. Here we report that although CypB deficiency resulted in lower lysine hydroxylation in the helical cross-linking sites, it was increased in the telopeptide cross-linking sites in tendon type I collagen. This resulted in a decrease in the lysine aldehyde-derived cross-links but generation of hydroxylysine aldehyde-derived cross-links. The latter were absent from the wild type and heterozygous mice. Glycosylation of hydroxylysine residues was moderately increased in the CypB null tendon. We found that CypB interacted with all lysyl hydroxylase isoforms (isoforms 1-3) and a putative lysyl hydroxylase-2 chaperone, 65-kDa FK506-binding protein. Tendon collagen in CypB null mice showed severe size and organizational abnormalities. The data indicate that CypB modulates collagen cross-linking by differentially affecting lysine hydroxylation in a site-specific manner, possibly via its interaction with lysyl hydroxylases and associated molecules. This study underscores the critical importance of collagen post-translational modifications in connective tissue formation. PMID:26934917

  16. Genetic analysis of L123 of the tRNA-mimicking eukaryote release factor eRF1, an amino acid residue critical for discrimination of stop codons

    PubMed Central

    Saito, Kazuki; Ito, Koichi

    2015-01-01

    In eukaryotes, the tRNA-mimicking polypeptide-chain release factor, eRF1, decodes stop codons on the ribosome in a complex with eRF3; this complex exhibits striking structural similarity to the tRNA–eEF1A–GTP complex. Although amino acid residues or motifs of eRF1 that are critical for stop codon discrimination have been identified, the details of the molecular mechanisms involved in the function of the ribosomal decoding site remain obscure. Here, we report analyses of the position-123 amino acid of eRF1 (L123 in Saccharomyces cerevisiae eRF1), a residue that is phylogenetically conserved among species with canonical and variant genetic codes. In vivo readthrough efficiency analysis and genetic growth complementation analysis of the residue-123 systematic mutants suggested that this amino acid functions in stop codon discrimination in a manner coupled with eRF3 binding, and distinctive from previously reported adjacent residues. Furthermore, aminoglycoside antibiotic sensitivity analysis and ribosomal docking modeling of eRF1 in a quasi-A/T state suggested a functional interaction between the side chain of L123 and ribosomal residues critical for codon recognition in the decoding site, as a molecular explanation for coupling with eRF3. Our results provide insights into the molecular mechanisms underlying stop codon discrimination by a tRNA-mimicking protein on the ribosome. PMID:25897120

  17. Identification, expression and antibacterial activities of an antimicrobial peptide NK-lysin from a marine fish Larimichthys crocea.

    PubMed

    Zhou, Qi-Jia; Wang, Jun; Liu, Min; Qiao, Ying; Hong, Wan-Shu; Su, Yong-Quan; Han, Kun-Huang; Ke, Qiao-Zhen; Zheng, Wei-Qiang

    2016-08-01

    As fundamental immunologic mechanism, the innate immunity system is more important than the specific immunity system in teleost fishes during pathogens infection. Antimicrobial peptides are integral parts of the innate immune system, and play significant roles against pathogens infection. NK-lysin, the compounds of the natural killer cells and cytotoxic T cells, are potent and effective antimicrobial peptides widely distributed in animals. In this study, we reported the sequence characteristics, expression profiles and antibacterial activities of a NK-lysin gene (Lc-NK-lysin) from a commercially important marine fish, the large yellow croaker (Larimichthys crocea). The open reading frame of Lc-NK-lysin cDNA sequence was 447 bp in length, coding 148 amino acids. The genomic DNA of Lc-NK-lysin has the common features of NK-lysin family, consisting of five exons and four introns, and in its deduced mature peptide, there are six well-conserved cysteine residues and a Saposin B domain. Lc-NK-lysin was expressed in all tested tissues (skin, muscle, gill, brain, head kidney, heart, liver, spleen, stomach and intestine) with different expression patterns. In pathogens infection the expression profiles of Lc-NK-lysin varied significantly in gill, head kidney, spleen and liver, indicating its role in immune response. Two peptides (Lc-NK-lysin-1 and Lc-NK-lysin-2) divided from the core region of the Lc-NK-lysin mature polypeptide were chemically synthesized and their antibacterial activities were examined; the potential function on the inhibition of bacteria propagation was revealed. Our results suggested that Lc-NK-lysin is a typical member of the NK-lysin family and as an immune-related gene it involves in the immune response when pathogens invasion. PMID:27238427

  18. Critical body-residues for lethal and sublethal effects of sediment-associated PAH on benthic copepods

    SciTech Connect

    Lotufo, G.R.

    1995-12-31

    Adult females of the meiobenthic copepod Schizopera knabeni were exposed to sediment-associated fluoranthene for 3, 6, 12, 24, 96, and 240 h. Sediment concentrations ranged from 25 to 1,661 nmol (5--336 {micro}g)/gdw and the TOC was 1.5%. Body burden increased to an apparent steady state after only 6 h. Elimination half-lives were 4.6 and 3.2 h in uncontaminated water and sediment, respectively. Toxic effects were only detected after 240 h as increased mortality and decreased offspring production. Significant mortality was observed only at the highest concentration; the LC50 was 1,011 nmol (204 {micro}g)/dgw. In contrast, offspring production was decreased at much lower concentrations, yielding an IC25 value of 148 nmol (30 {micro}g)/dgw. Lethal critical body residue (CBR) was determined as a 10-d LD50 of 15.5 {micro}mol/g dry tissue. By measuring PAH concentrations in the body and eggs of females, CBRs for reproductive output were determined as IC25 values of 2 and 3.1 {micro}mol/gdw, respectively. PAH sublethal effects on feeding rate were also investigated Adult copepods were exposed to {sup 14}C sediment-associated fluoranthene for 24 h were fed {sup 3}H-labeled algae for 3 h. Ingestion rate was significantly decreased at tissue concentrations as low as 1 {micro}mol/gdw and yielded an IC25 value of 0.6 {micro}mol/gdw. Similar findings were obtained using another species of estuarine copepod, Coullana sp. Non-polar narcotic compounds such as PAH cause a nonspecific disturbance of the functioning of cell membrane which results in decreased overall activity. Measurement of CBR associated with decreased feeding is proposed as a direct method to quantify sublethal narcotizing effects of organic compounds.

  19. Protein lysine methylation by seven-β-strand methyltransferases.

    PubMed

    Falnes, Pål Ø; Jakobsson, Magnus E; Davydova, Erna; Ho, Angela; Małecki, Jędrzej

    2016-07-15

    Methylation of biomolecules is a frequent biochemical reaction within the cell, and a plethora of highly specific methyltransferases (MTases) catalyse the transfer of a methyl group from S-adenosylmethionine (AdoMet) to various substrates. The posttranslational methylation of lysine residues, catalysed by numerous lysine (K)-specific protein MTases (KMTs), is a very common and important protein modification, which recently has been subject to intense studies, particularly in the case of histone proteins. The majority of KMTs belong to a class of MTases that share a defining 'SET domain', and these enzymes mostly target lysines in the flexible tails of histones. However, the so-called seven-β-strand (7BS) MTases, characterized by a twisted beta-sheet structure and certain conserved sequence motifs, represent the largest MTase class, and these enzymes methylate a wide range of substrates, including small metabolites, lipids, nucleic acids and proteins. Until recently, the histone-specific Dot1/DOT1L was the only identified eukaryotic 7BS KMT. However, a number of novel 7BS KMTs have now been discovered, and, in particular, several recently characterized human and yeast members of MTase family 16 (MTF16) have been found to methylate lysines in non-histone proteins. Here, we review the status and recent progress on the 7BS KMTs, and discuss these enzymes at the levels of sequence/structure, catalytic mechanism, substrate recognition and biological significance. PMID:27407169

  20. Identification of three critical acidic residues of poly(ADP-ribose) glycohydrolase involved in catalysis: determining the PARG catalytic domain

    PubMed Central

    Patel, Chandra N.; Koh, David W.; Jacobson, Myron K.; Oliveira, Marcos A.

    2005-01-01

    PARG [poly(ADP-ribose) glycohydrolase] catalyses the hydrolysis of α(1″→2′) or α(1‴→2″) O-glycosidic linkages of ADP-ribose polymers to produce free ADP-ribose. We investigated possible mechanistic similarities between PARG and glycosidases, which also cleave O-glycosidic linkages. Glycosidases typically utilize two acidic residues for catalysis, thus we targeted acidic residues within a conserved region of bovine PARG that has been shown to contain an inhibitor-binding site. The targeted glutamate and aspartate residues were changed to asparagine in order to minimize structural alterations. Mutants were purified and assayed for catalytic activity, as well as binding, to an immobilized PARG inhibitor to determine ability to recognize substrate. Our investigation revealed residues essential for PARG catalytic activity. Two adjacent glutamic acid residues are found in the conserved sequence Gln755-Glu-Glu757, and a third residue found in the conserved sequence Val737-Asp-Phe-Ala-Asn741. Our functional characterization of PARG residues, along with recent identification of an inhibitor-binding residue Tyr796 and a glycine-rich region Gly745-Gly-Gly747 important for PARG function, allowed us to define a PARG ‘signature sequence’ [vDFA-X3-GGg-X6–8-vQEEIRF-X3-PE-X14-E-X12-YTGYa], which we used to identify putative PARG sequences across a range of organisms. Sequence alignments, along with our mapping of PARG functional residues, suggest the presence of a conserved catalytic domain of approx. 185 residues which spans residues 610–795 in bovine PARG. PMID:15658938

  1. Prion Protein Prolines 102 and 105 and the Surrounding Lysine Cluster Impede Amyloid Formation.

    PubMed

    Kraus, Allison; Anson, Kelsie J; Raymond, Lynne D; Martens, Craig; Groveman, Bradley R; Dorward, David W; Caughey, Byron

    2015-08-28

    Human prion diseases can have acquired, sporadic, or genetic origins, each of which results in the conversion of prion protein (PrP) to transmissible, pathological forms. The genetic prion disease Gerstmann-Straussler-Scheinker syndrome can arise from point mutations of prolines 102 or 105. However, the structural effects of these two prolines, and mutations thereof, on PrP misfolding are not well understood. Here, we provide evidence that individual mutations of Pro-102 or Pro-105 to noncyclic aliphatic residues such as the Gerstmann-Straussler-Scheinker-linked leucines can promote the in vitro formation of PrP amyloid with extended protease-resistant cores reminiscent of infectious prions. This effect was enhanced by additional charge-neutralizing mutations of four nearby lysine residues comprising the so-called central lysine cluster. Substitution of these proline and lysine residues accelerated PrP conversion such that spontaneous amyloid formation was no longer slower than scrapie-seeded amyloid formation. Thus, Pro-102 and Pro-105, as well as the lysines in the central lysine cluster, impede amyloid formation by PrP, implicating these residues as key structural modulators in the conversion of PrP to disease-associated types of amyloid. PMID:26175152

  2. Differences in Lysine pKa Values May Be Used to Improve NMR Signal Dispersion in Reductively Methylated Proteins

    PubMed Central

    Abraham, Sherwin J.; Kobayashi, Tomoyoshi; Solaro, R. John; Gaponenko, Vadim

    2009-01-01

    Summary Reductive methylation of lysine residues in proteins offers a way to introduce 13C methyl groups into otherwise unlabeled molecules. The 13C methyl groups on lysines possess favorable relaxation properties that allow highly sensitive NMR signal detection. One of the major limitations in the use of reductive methylation in NMR is the signal overlap of 13C methyl groups in NMR spectra. Here we show that the uniform influence of the solvent on chemical shifts of exposed lysine methyl groups could be overcome by adjusting the pH of the buffering solution closer to the pKa of lysine side chains. Under these conditions, due to variable pKa values of individual lysine side chains in the protein of interest different levels of lysine protonation are observed. These differences are reflected in the chemical shift differences of methyl groups in reductively methylated lysines. We show that this approach is successful in four different proteins including Ca2+-bound Calmodulin, Lysozyme, Ca2+-bound Troponin C, and Glutathione S-Transferase. In all cases significant improvement in NMR spectral resolution of methyl signals in reductively methylated proteins was obtained. The increased spectral resolution helps with more precise characterization of protein structural rearrangements caused by ligand binding as shown by studying binding of Calmodulin antagonist trifluoperazine to Calmodulin. Thus, this approach may be used to increase resolution in NMR spectra of 13C methyl groups on lysine residues in reductively methylated proteins that enhances the accuracy of protein structural assessment. PMID:19280122

  3. Use of acetimidation in the NMR identification of neurophysin lysine protons

    SciTech Connect

    Sardana, V.; Breslow, E.

    1986-05-01

    Acetimidation of the two lysine residues of neurophysin (NP) results in localized changes in the proton magnetic resonance spectrum, allowing identification of lysine side-chain resonances. Neither peptide-binding nor protein self-association appeared to be significantly altered by acetimidation. Additionally, no significant effect of either peptide-binding or self-association on lysine epsilon-CH/sub 2/ protons was seen. However, dimerization-induced NMR changes in the 1.6-1.8 ppm region, associated with lysine ..beta..,..gamma..,sigma protons, were altered in the acetimidated protein. In particular, while the spectrum of the acetimidated NP monomer was almost identical to that of the native protein, a shoulder at 1.72 ppm in the native protein dimer was shifted upfield in the modified dimer. Additionally the direction of NMR shifts in the 1.6-1.8 ppm region normally associated with peptide binding to the NP dimer appeared to be reversed in the acetimidated protein. Binding-induced and dimerization-induced changes in all other regions of the spectrum were identical in the native and modified proteins. These results suggest that one or both NP lysine residues may be near the dimer subunit interface and indicate an effect of peptide-binding on lysine side-chain environment.

  4. BIOLOGICAL ADHESIVES. Adaptive synergy between catechol and lysine promotes wet adhesion by surface salt displacement.

    PubMed

    Maier, Greg P; Rapp, Michael V; Waite, J Herbert; Israelachvili, Jacob N; Butler, Alison

    2015-08-01

    In physiological fluids and seawater, adhesion of synthetic polymers to solid surfaces is severely limited by high salt, pH, and hydration, yet these conditions have not deterred the evolution of effective adhesion by mussels. Mussel foot proteins provide insights about adhesive adaptations: Notably, the abundance and proximity of catecholic Dopa (3,4-dihydroxyphenylalanine) and lysine residues hint at a synergistic interplay in adhesion. Certain siderophores—bacterial iron chelators—consist of paired catechol and lysine functionalities, thereby providing a convenient experimental platform to explore molecular synergies in bioadhesion. These siderophores and synthetic analogs exhibit robust adhesion energies (E(ad) ≥-15 millijoules per square meter) to mica in saline pH 3.5 to 7.5 and resist oxidation. The adjacent catechol-lysine placement provides a "one-two punch," whereby lysine evicts hydrated cations from the mineral surface, allowing catechol binding to underlying oxides. PMID:26250681

  5. Adaptive synergy between catechol and lysine promotes wet adhesion by surface salt displacement

    NASA Astrophysics Data System (ADS)

    Maier, Greg P.; Rapp, Michael V.; Waite, J. Herbert; Israelachvili, Jacob N.; Butler, Alison

    2015-08-01

    In physiological fluids and seawater, adhesion of synthetic polymers to solid surfaces is severely limited by high salt, pH, and hydration, yet these conditions have not deterred the evolution of effective adhesion by mussels. Mussel foot proteins provide insights about adhesive adaptations: Notably, the abundance and proximity of catecholic Dopa (3,4-dihydroxyphenylalanine) and lysine residues hint at a synergistic interplay in adhesion. Certain siderophores—bacterial iron chelators—consist of paired catechol and lysine functionalities, thereby providing a convenient experimental platform to explore molecular synergies in bioadhesion. These siderophores and synthetic analogs exhibit robust adhesion energies (Ead ≥-15 millijoules per square meter) to mica in saline pH 3.5 to 7.5 and resist oxidation. The adjacent catechol-lysine placement provides a “one-two punch,” whereby lysine evicts hydrated cations from the mineral surface, allowing catechol binding to underlying oxides.

  6. Structure of the DNA Ligase-Adenylate Intermediate: Lysine (ε-amino)-Linked Adenosine Monophosphoramidate*

    PubMed Central

    Gumport, Richard I.; Lehman, I. R.

    1971-01-01

    Proteolytic degradation of the Escherichia coli DNA ligase-adenylate intermediate releases adenosine 5′-monophosphate linked to the ε-amino group of lysine by a phosphoamide bond. Measurements of the rate of hydroxylaminolysis of the ligase-adenylate provide further support for a phosphoamide linkage in the native enzyme. Lysine (ε-amino)-linked adenosine monophosphoramidate has also been isolated from the T4 phage-induced ligase-adenylate intermediate. These results indicate that an initial step of the DNA ligase reaction consists of the nucleophilic attack of the ε-amino group of a lysine residue of the enzyme on the adenylyl phosphorus of DPN or ATP that leads to the formation of enzyme-bound lysine (εamino)-linked adenosine monophosphoramidate. PMID:4944632

  7. Acetylation of Surface Lysine Groups of a Protein Alters the Organization and Composition of Its Crystal Contacts.

    PubMed

    Kang, Kyungtae; Choi, Jeong-Mo; Fox, Jerome M; Snyder, Phillip W; Moustakas, Demetri T; Whitesides, George M

    2016-07-14

    This paper uses crystals of bovine carbonic anhydrase (CA) and its acetylated variant to examine (i) how a large negative formal charge can be accommodated in protein-protein interfaces, (ii) why lysine residues are often excluded from them, and (iii) how changes in the surface charge of a protein can alter the structure and organization of protein-protein interfaces. It demonstrates that acetylation of lysine residues on the surface of CA increases the participation of polar residues (particularly acetylated lysine) in protein-protein interfaces, and decreases the participation of nonpolar residues in those interfaces. Negatively charged residues are accommodated in protein-protein interfaces via (i) hydrogen bonds or van der Waals interactions with polar residues or (ii) salt bridges with other charged residues. The participation of acetylated lysine in protein-protein interfaces suggests that unacetylated lysine tends to be excluded from interfaces because of its positive charge, and not because of a loss in conformational entropy. Results also indicate that crystal contacts in acetylated CA become less constrained geometrically and, as a result, more closely packed (i.e., more tightly clustered spatially) than those of native CA. This study demonstrates a physical-organic approach-and a well-defined model system-for studying the role of charges in protein-protein interactions. PMID:27292012

  8. Nonhemolytic Cell-Penetrating Peptides: Site Specific Introduction of Glutamine and Lysine Residues into the α-Helical Peptide Causes Deletion of Its Direct Membrane Disrupting Ability but Retention of Its Cell Penetrating Ability.

    PubMed

    Kim, Seoyeon; Hyun, Soonsil; Lee, Yuri; Lee, Yan; Yu, Jaehoon

    2016-09-12

    Cell-penetrating peptides (CPPs) often have cationic and amphipathic characteristics that are commonly associated with α-helical peptides. These features give CPPs both membrane demolishing and penetrating abilities. To make CPPs safe for biomedical applications, their toxicities resulting from their membrane demolishing abilities must be removed while their cell penetrating abilities must be retained. In this study, we systematically constructed mutants of the amphipathic α-helical model peptide (LKKLLKLLKKLLKLAG, LK peptide). The hydrophobic amino acid leucine in the LK peptide was replaced with hydrophilic amino acids to reduce hemolytic or cell toxicity. Most of the mutants were found to have weakened membrane disrupting abilities, but their cell penetrating abilities were also weakened. However, the L8Q and L8K mutants were found to have low micromolar range cell penetrating ability and almost no membrane disrupting ability. These selected mutants utilize energy-dependent endocytosis mechanisms instead of an energy-independent direct cell penetrating mechanism to enter cells. In addition, the mutants can be used to deliver the anticancer drug methotrexate (MTX) to cells, thereby overcoming resistance to this drug. To determine if the effect of these mutations on the membrane disrupting and cell penetrating abilities is general, Q and K mutations of the natural amphipathic α-helical antimicrobial peptide (AMP), LL37, were introduced. Specific positional Q and K mutants of LL37 were found to have lower hemolytic toxicities and preserved the ability to penetrate eukaryotic cells such as MDA-MB-231 cells. Taken together, observations made in this work suggest that interrupting the global hydrophobicity of amphipathic α-helical CPPs and AMPs, by replacing hydrophobic residues with mildly hydrophilic amino acids such as Q and K, might be an ideal strategy for constructing peptides that have strong cell penetrating abilities and weak cell membrane disrupting

  9. Adding a Lysine Mimic in the Design of Potent Inhibitors of Histone Lysine Methyltransferases

    SciTech Connect

    Chang, Yanqi; Ganesh, Thota; Horton, John R.; Spannhoff, Astrid; Liu, Jin; Sun, Aiming; Zhang, Xing; Bedford, Mark T.; Shinkai, Yoichi; Snyder, James P.; Cheng, Xiaodong

    2010-07-19

    Dynamic histone lysine methylation involves the activities of modifying enzymes (writers), enzymes removing modifications (erasers), and readers of the histone code. One common feature of these activities is the recognition of lysines in methylated and unmethylated states, whether they are substrates, reaction products, or binding partners. We applied the concept of adding a lysine mimic to an established inhibitor (BIX-01294) of histone H3 lysine 9 methyltransferases G9a and G9a-like protein by including a 5-aminopentyloxy moiety, which is inserted into the target lysine-binding channel and becomes methylated by G9a-like protein, albeit slowly. The compound enhances its potency in vitro and reduces cell toxicity in vivo. We suggest that adding a lysine or methyl-lysine mimic should be considered in the design of small-molecule inhibitors for other methyl-lysine writers, erasers, and readers.

  10. Identification of residues in the hepatitis C virus core protein that are critical for capsid assembly in a cell-free system.

    PubMed

    Klein, Kevin C; Dellos, Sheri R; Lingappa, Jaisri R

    2005-06-01

    Significant advances have been made in understanding hepatitis C virus (HCV) replication through development of replicon systems. However, neither replicon systems nor standard cell culture systems support significant assembly of HCV capsids, leaving a large gap in our knowledge of HCV virion formation. Recently, we established a cell-free system in which over 60% of full-length HCV core protein synthesized de novo in cell extracts assembles into HCV capsids by biochemical and morphological criteria. Here we used mutational analysis to identify residues in HCV core that are important for capsid assembly in this highly reproducible cell-free system. We found that basic residues present in two clusters within the N-terminal 68 amino acids of HCV core played a critical role, while the uncharged linker domain between them was not. Furthermore, the aspartate at position 111, the region spanning amino acids 82 to 102, and three serines that are thought to be sites of phosphorylation do not appear to be critical for HCV capsid formation in this system. Mutation of prolines important for targeting of core to lipid droplets also failed to alter HCV capsid assembly in the cell-free system. In addition, wild-type HCV core did not rescue assembly-defective mutants. These data constitute the first systematic and quantitative analysis of the roles of specific residues and domains of HCV core in capsid formation. PMID:15890921

  11. Quantitative Assessment of Protein Interaction with Methyl-Lysine Analogues by Hybrid Computational and Experimental Approaches

    PubMed Central

    2011-01-01

    In cases where binding ligands of proteins are not easily available, structural analogues are often used. For example, in the analysis of proteins recognizing different methyl-lysine residues in histones, methyl-lysine analogues based on methyl-amino-alkylated cysteine residues have been introduced. Whether these are close enough to justify quantitative interpretation of binding experiments is however questionable. To systematically address this issue, we developed, applied, and assessed a hybrid computational/experimental approach that extracts the binding free energy difference between the native ligand (methyl-lysine) and the analogue (methyl-amino-alkylated cysteine) from a thermodynamic cycle. Our results indicate that measured and calculated binding differences are in very good agreement and therefore allow the correction of measured affinities of the analogues. We suggest that quantitative binding parameters for defined ligands in general can be derived by this method with remarkable accuracy. PMID:21991995

  12. N-formylation of lysine in histone proteins as a secondary modification arising from oxidative DNA damage

    PubMed Central

    Jiang, Tao; Zhou, Xinfeng; Taghizadeh, Koli; Dong, Min; Dedon, Peter C.

    2007-01-01

    The posttranslational modification of histone and other chromatin proteins has a well recognized but poorly defined role in the physiology of gene expression. With implications for interfering with these epigenetic mechanisms, we now report the existence of a relatively abundant secondary modification of chromatin proteins, the N6-formylation of lysine that appears to be uniquely associated with histone and other nuclear proteins. Using both radiolabeling and sensitive bioanalytical methods, we demonstrate that the formyl moiety of 3′-formylphosphate residues arising from 5′-oxidation of deoxyribose in DNA, caused by the enediyne neocarzinostatin, for example, acylate the N6-amino groups of lysine side chains. A liquid chromatography (LC)–tandem mass spectrometry (MS) method was developed to quantify the resulting N6-formyl-lysine residues, which were observed to be present in unperturbed cells and all sources of histone proteins to the extent of 0.04–0.1% of all lysines in acid-soluble chromatin proteins including histones. Cells treated with neocarzinostatin showed a clear dose–response relationship for the formation of N6-formyl-lysine, with this nucleosome linker-selective DNA-cleaving agent causing selective N6-formylation of the linker histone H1. The N6-formyl-lysine residue appears to represent an endogenous histone secondary modification, one that bears chemical similarity to lysine N6-acetylation recognized as an important determinant of gene expression in mammalian cells. The N6-formyl modification of lysine may interfere with the signaling functions of lysine acetylation and methylation and thus contribute to the pathophysiology of oxidative and nitrosative stress. PMID:17190813

  13. Hemoglobin Labeled by Radioactive Lysine

    DOE R&D Accomplishments Database

    Bale, W. F.; Yuile, C. L.; DeLaVergne, L.; Miller, L. L.; Whipple, G. H.

    1949-12-08

    This paper reports on the utilization of tagged epsilon carbon of DL-lysine by a dog both anemic and hypoproteinemic due to repeated bleeding plus a diet low in protein. The experiment extended over period of 234 days, a time sufficient to indicate an erythrocyte life span of at least 115 days based upon the rate of replacement of labeled red cell proteins. The proteins of broken down red cells seem not to be used with any great preference for the synthesis of new hemoglobin.

  14. Nε−Lysine Acetylation of a Bacterial Transcription Factor Inhibits Its DNA-Binding Activity

    PubMed Central

    Thao, Sandy; Chen, Chien-Sheng; Zhu, Heng; Escalante-Semerena, Jorge C.

    2010-01-01

    Evidence suggesting that eukaryotes and archaea use reversible Nε-lysine (Nε-Lys) acetylation to modulate gene expression has been reported, but evidence for bacterial use of Nε-Lys acetylation for this purpose is lacking. Here, we report data in support of the notion that bacteria can control gene expression by modulating the acetylation state of transcription factors (TFs). We screened the E. coli proteome for substrates of the bacterial Gcn5-like protein acetyltransferase (Pat). Pat acetylated four TFs, including the RcsB global regulatory protein, which controls cell division, and capsule and flagellum biosynthesis in many bacteria. Pat acetylated residue Lys180 of RcsB, and the NAD+-dependent Sir2 (sirtuin)-like protein deacetylase (CobB) deacetylated acetylated RcsB (RcsBAc), demonstrating that Nε-Lys acetylation of RcsB is reversible. Analysis of RcsBAc and variant RcsB proteins carrying substitutions at Lys180 provided biochemical and physiological evidence implicating Lys180 as a critical residue for RcsB DNA-binding activity. These findings further the likelihood that reversible Nε-Lys acetylation of transcription factors is a mode of regulation of gene expression used by all cells. PMID:21217812

  15. Favored and disfavored pathways of protein crosslinking by glucose: glucose lysine dimer (GLUCOLD) and crossline versus glucosepane

    PubMed Central

    Nemet, Ina; Strauch, Christopher M.

    2010-01-01

    We describe the isolation and molecular characterization of a novel glucose-lysine dimer crosslink 1,3-bis-(5-amino-5-carboxypentyl)-4-(1′,2′,3′,4′-tetrahydroxybutyl)-3H-imidazolium salt, named GLUCOLD. GLUCOLD was easily formed from the Amadori product (fructose–lysine). However, when BSA was incubated with 100 mM glucose for 25 days, the levels of the lysine-lysine glucose crosslinks GLUCOLD and CROSSLINE were only 21 and <1 pmol/mg, respectively, compared to 611 pmol/mg protein for the lysine-arginine GLUCOSEPANE crosslink, in spite of more than 20 potential lysine-lysine crosslinking sites in the protein. Mechanistic investigation revealed that metal-free phosphate ions catalyzed formation of fructose–lysine and all three crosslinks from amino acids, while cationic MOPS buffer had an opposite effect. This together with the rapid formation of N6-1,4-dideoxy-5,6-dioxoglucosone derivatives by dicarbonyl trapping agents, such as 1,2-diaminobenzene or γ-guanidinobutyric acid, strongly suggests that enolization of the Amadori product and trapping of the 5,6-dioxo derivative by arginine residues constitutes the major pathway for glucose-mediated crosslinking in proteins. PMID:20607325

  16. Favored and disfavored pathways of protein crosslinking by glucose: glucose lysine dimer (GLUCOLD) and crossline versus glucosepane.

    PubMed

    Nemet, Ina; Strauch, Christopher M; Monnier, Vincent M

    2011-01-01

    We describe the isolation and molecular characterization of a novel glucose-lysine dimer crosslink 1,3-bis-(5-amino-5-carboxypentyl)-4-(1',2',3',4'-tetrahydroxybutyl)-3H-imidazolium salt, named GLUCOLD. GLUCOLD was easily formed from the Amadori product (fructose-lysine). However, when BSA was incubated with 100 mM glucose for 25 days, the levels of the lysine-lysine glucose crosslinks GLUCOLD and CROSSLINE were only 21 and <1 pmol/mg, respectively, compared to 611 pmol/mg protein for the lysine-arginine GLUCOSEPANE crosslink, in spite of more than 20 potential lysine-lysine crosslinking sites in the protein. Mechanistic investigation revealed that metal-free phosphate ions catalyzed formation of fructose-lysine and all three crosslinks from amino acids, while cationic MOPS buffer had an opposite effect. This together with the rapid formation of N (6)-1,4-dideoxy-5,6-dioxoglucosone derivatives by dicarbonyl trapping agents, such as 1,2-diaminobenzene or γ-guanidinobutyric acid, strongly suggests that enolization of the Amadori product and trapping of the 5,6-dioxo derivative by arginine residues constitutes the major pathway for glucose-mediated crosslinking in proteins. PMID:20607325

  17. Identification of basic residues in RAG2 critical for DNA binding by the RAG1-RAG2 complex.

    PubMed

    Fugmann, S D; Schatz, D G

    2001-10-01

    In V(D)J recombination, the RAG1 and RAG2 proteins are the essential components of the complex that catalyzes DNA cleavage. RAG1 has been shown to play a central role in DNA binding and catalysis. In contrast, the molecular roles of RAG2 in V(D)J recombination are unknown. To address this, we individually mutated 36 evolutionarily conserved basic and hydroxy group containing residues within RAG2. Biochemical analysis of the recombinant RAG2 proteins led to the identification of a number of basic residue mutants defective in catalysis in vitro and V(D)J recombination in vivo. Five of these were deficient in binding of the RAG1-RAG2 complex to its cognate DNA target sequence while interacting normally with RAG1. Our findings provide support for the direct involvement of RAG2 in DNA binding during all steps of the cleavage reaction. PMID:11684024

  18. Effect of pyruvate dehydrogenase complex deficiency on L-lysine production with Corynebacterium glutamicum.

    PubMed

    Blombach, Bastian; Schreiner, Mark E; Moch, Matthias; Oldiges, Marco; Eikmanns, Bernhard J

    2007-09-01

    Intracellular precursor supply is a critical factor for amino acid productivity of Corynebacterium glutamicum. To test for the effect of improved pyruvate availability on L-lysine production, we deleted the aceE gene encoding the E1p enzyme of the pyruvate dehydrogenase complex (PDHC) in the L-lysine-producer C. glutamicum DM1729 and characterised the resulting strain DM1729-BB1 for growth and L-lysine production. Compared to the host strain, C. glutamicum DM1729-BB1 showed no PDHC activity, was acetate auxotrophic and, after complete consumption of the available carbon sources glucose and acetate, showed a more than 50% lower substrate-specific biomass yield (0.14 vs 0.33 mol C/mol C), an about fourfold higher biomass-specific L-lysine yield (5.27 vs 1.23 mmol/g cell dry weight) and a more than 40% higher substrate-specific L-lysine yield (0.13 vs 0.09 mol C/mol C). Overexpression of the pyruvate carboxylase or diaminopimelate dehydrogenase genes in C. glutamicum DM1729-BB1 resulted in a further increase in the biomass-specific L-lysine yield by 6 and 56%, respectively. In addition to L-lysine, significant amounts of pyruvate, L-alanine and L-valine were produced by C. glutamicum DM1729-BB1 and its derivatives, suggesting a surplus of precursor availability and a further potential to improve L-lysine production by engineering the L-lysine biosynthetic pathway. PMID:17333167

  19. Lysine-scanning Mutagenesis Reveals an Amendable Face of the Cyclotide Kalata B1 for the Optimization of Nematocidal Activity*

    PubMed Central

    Huang, Yen-Hua; Colgrave, Michelle L.; Clark, Richard J.; Kotze, Andrew C.; Craik, David J.

    2010-01-01

    Cyclotides are a family of macrocyclic peptides that combine the unique features of a head-to-tail cyclic backbone and a cystine knot motif, the combination of which imparts them with extraordinary stability. The prototypic cyclotide kalata B1 is toxic against two economically important gastrointestinal nematode parasites of sheep, Haemonchus contortus and Trichostrongylus colubriformis. A lysine scan was conducted to examine the effect of the incorporation of positive charges into the kalata B1 cyclotide framework. Each of the non-cysteine residues in this 29-amino acid peptide was successively substituted with lysine, and the nematocidal and hemolytic activities of the suite of mutants were determined. Substitution of 11 residues within kalata B1 decreased the nematocidal activity dramatically. On the other hand, six other residues that are clustered on the surface of kalata B1 were tolerant to Lys substitution, and indeed the introduction of positively charged residues into this region increased nematocidal activity. This activity was increased further in double and triple lysine mutants, with a maximal increase (relative to the native kalata B1) of 13-fold obtained with a triple lysine mutant (mutated at positions Thr-20, Asn-29, and Gly-1). Hemolytic activity correlated with the nematocidal activity of all lysine mutants. Our data clearly highlight the residues crucial for nematocidal and hemolytic activity in cyclotides, and demonstrate that the nematocidal activity of cyclotides can be increased by incorporation of basic amino acids. PMID:20103593

  20. Identification of Lysine Acetylation in Mycobacterium abscessus Using LC-MS/MS after Immunoprecipitation.

    PubMed

    Guo, Jintao; Wang, Changwei; Han, Yi; Liu, Zhiyong; Wu, Tian; Liu, Yan; Liu, Yang; Tan, Yaoju; Cai, Xinshan; Cao, Yuanyuan; Wang, Bangxing; Zhang, Buchang; Liu, Chunping; Tan, Shouyong; Zhang, Tianyu

    2016-08-01

    Mycobacterium abscessus (MAB), which manifests in the pulmonary system, is one of the neglected causes of nontuberculous mycobacteria (NTM) infection. Treatment against MAB is difficult, characterized by its intrinsic antibiotic drug resistance. Lysine acetylation can alter the physiochemical property of proteins in living organisms. This study aimed to determine if this protein post-translational modification (PTM) exists in a clinical isolate M. abscessus GZ002. We used the antiacetyl-lysine immunoprecipitation to enrich the low-abundant PTM proteins, followed by the LC-MS/MS analysis. The lysine acetylome of M. abscessus GZ002 was determined. There were 459 lysine acetylation sites found in 289 acetylated proteins. Lysine acetylation occurred in 5.87% of the M. abscessus GZ002 proteome, and at least 25% of them were growth essential. Aerobic respiration and carbohydrate metabolic pathways of M. abscessus GZ002 were enriched with lysine acetylation. Through bioinformatics analysis, we identified four major acetyl motif logos (K(ac)Y, K(ac)F, K(ac)H, and DK(ac)). Further comparison of the reported M. tuberculosis (MTB) acetylomes and that of MAB GZ002 revealed several common features between these two species. The lysine residues of several antibiotic-resistance, virulence, and persistence-related proteins were acetylated in both MAB GZ002 and MTB. There were 51 identical acetylation sites in 37 proteins found in common between MAB GZ002 and MTB. Overall, we demonstrate a profile of lysine acetylation in MAB GZ002 proteome that shares similarities with MTB. Interventions that target at these conserved sections may be valuable as anti-NTM or anti-TB therapies. PMID:27323652

  1. Molecular Pathways: Deregulation of Histone 3 Lysine 27 Methylation in Cancer—Different Paths, Same Destination

    PubMed Central

    Ezponda, Teresa; Licht, Jonathan D.

    2014-01-01

    Methylation of lysine 27 on histone 3 (H3K27me), a modification associated with gene repression, plays a critical role in regulating the expression of genes that determine the balance between cell differentiation and proliferation. Alteration of the level of this histone modification has emerged as a recurrent theme in many types of cancer, demonstrating that either excess or lack of H3K27 methylation can have oncogenic effects. Cancer genome sequencing has revealed the genetic basis of H3K27me deregulation, including mutations of the components of the H3K27 methyltransferase complex PRC2 and accessory proteins, and deletions and inactivating mutations of the H3K27 demethylase UTX in a wide variety of neoplasms. More recently, mutations of lysine 27 on histone 3 itself were shown to prevent H3K27me in pediatric glioblastomas. Aberrant expression or mutations in proteins that recognize H3K27me3 also occur in cancer and may result in misinterpretation of this mark. Additionally, due to the crosstalk between different epigenetic modifications, alterations of chromatin modifiers controlling H3K36me, or even mutations of this residue, can ultimately regulate H3K27me levels and distribution across the genome. The significance of mutations altering H3K27me is underscored by the fact that many tumors harboring such lesions often have a poor clinical outcome. New therapeutic approaches targeting aberrant H3K27 methylation include small molecules that block the action of mutant EZH2 in germinal center-derived lymphoma. Understanding the biological consequences and gene expression pathways affected by aberrant H3K27 methylation may also lead to other new therapeutic strategies. PMID:24987060

  2. Critical regions and residues for self-interaction of grapevine leafroll-associated virus 2 protein p24.

    PubMed

    Liu, Qing; Guo, Ran; Li, Mingjun; Feng, Ming; Wang, Xianyou; Wang, Qi; Cheng, Yuqin

    2016-07-15

    The 24-kDa protein (p24) encoded by grapevine leafroll-associated virus 2 (GLRaV-2) is an RNA-silencing suppressor. In this work, a yeast two-hybrid system (YTHS) and bimolecular fluorescence complementation analyses showed that GLRaV-2 p24 can interact with itself, and that this interaction occurs in the cytoplasm of Nicotiana benthamiana cells. To identify the functional region(s) and crucial amino acid residues required for p24 self-interaction, various truncated and substitution mutants were generated. YTHS assay showed that in both homologous pairing and pairing with the wild-type p24, the functional regions mapped to aa 10-180 or 1-170 which contain, respectively, all seven α-helices or the first six α-helices and the N-terminal end (aa 1-9) of the protein. When only the full-length p24 was an interaction partner, the functional region of aa 1-170 could be further mapped to aa 1-140 which contains four α-helices plus most of the fifth α-helix. Further analysis with substitution mutants demonstrated that hydrophobic residues I35/F38/V85/V89/W149 and V162/L169/L170, which may, respectively, mediate the inter-domain interaction of the same p24 monomer and the tail-to-tail association between two p24 counterparts, are crucial for homotypic p24-p24 interaction. In addition, substitution of two basic residues-R2 or R86-of p24, which may play important functional roles in RNA binding, did not seem to affect self-interaction of the mutants in yeast but had obvious effects in plant cells. Taken together, our results demonstrate the functional regions and crucial amino acids for p24 self-interaction. PMID:27084306

  3. Amino acid residues in the Ler protein critical for derepression of the LEE5 promoter in enteropathogenic E. coli.

    PubMed

    Choi, Su-Mi; Jeong, Jae-Ho; Choy, Hyon E; Shin, Minsang

    2016-08-01

    Enteropathogenic E. coli causes attaching and effacing (A/E) intestinal lesions. The genes involved in the formation of A/E lesions are encoded within a chromosomal island comprising of five major operons, LEE1-5. The global regulator H-NS represses the expression of these operons. Ler, a H-NS homologue, counteracts the H-NS-mediated repression. Using a novel genetic approach, we identified the amino acid residues in Ler that are involved in the interaction with H-NS: I20 and L23 in the C-terminal portion of α-helix 3, and I42 in the following unstructured linker region. PMID:27480636

  4. Engineering a Lysine-ON Riboswitch for Metabolic Control of Lysine Production in Corynebacterium glutamicum.

    PubMed

    Zhou, Li-Bang; Zeng, An-Ping

    2015-12-18

    Riboswitches are natural RNA elements that regulate gene expression by binding a ligand. Here, we demonstrate the possibility of altering a natural lysine-OFF riboswitch from Eschericia coli (ECRS) to a synthetic lysine-ON riboswitch and using it for metabolic control. To this end, a lysine-ON riboswitch library was constructed using tetA-based dual genetic selection. After screening the library, the functionality of the selected lysine-ON riboswitches was examined using a report gene, lacZ. Selected lysine-ON riboswitches were introduced into the lysE gene (encoding a lysine transport protein) of Corynebacterium glutamicum and used to achieve dynamic control of lysine transport in a recombinant lysine-producing strain, C. glutamicum LPECRS, which bears a deregulated aspartokinase and a lysine-OFF riboswitch for dynamic control of the enzyme citrate synthase. Batch fermentation results of the strains showed that the C. glutamicum LPECRS strain with an additional lysine-ON riboswitch for the control of lysE achieved a 21% increase in the yield of lysine compared to that of the C. glutamicum LPECRS strain and even a 89% increase in yield compared to that of the strain with deregulated aspartokinase. This work provides a useful approach to generate lysine-ON riboswitches for C. glutamicum metabolic engineering and demonstrates for the first time a synergetic effect of lysine-ON and -OFF riboswitches for improving lysine production in this industrially important microorganism. The approach can be used to dynamically control other genes and can be applied to other microorganisms. PMID:26300047

  5. Distance Restraints from Crosslinking Mass Spectrometry: Mining a Molecular Dynamics Simulation Database to Evaluate Lysine-Lysine Distances

    SciTech Connect

    Merkley, Eric D.; Rysavy, Steven; Kahraman, Abdullah; Hafen, Ryan P.; Daggett, Valerie; Adkins, Joshua N.

    2014-03-18

    Integrative structural biology models the structures of protein complexes that are intractable by classical structural methods (because of extreme size, dynamics, or heterogeneity) by combining computational structural modeling with data from experimental methods. One such method is chemical cross-linking mass spectrometry (XL-MS), in which cross-linked peptides, derived from a covalently cross-linked protein complex and identified by liquid chromatography-mass spectrometry, pinpoint protein residues close in three-dimensional space. The commonly used lysine-reactive N-hydroxysuccinimide ester reagents disuccinimidylsuberate (DSS) and bis(sulfosuccinimidyl)suberate (BS3) have a linker arm that is 11.4 Å long when fully extended. However, XL-MS studies on proteins of known structure frequently report cross-links that exceed this distance. Typically, a tolerance of ~3 Å is added to the theoretical maximum to account for this observation, with little justification for the value chosen. We used the Dynameomics database, a repository of high-quality molecular dynamics simulations of 807 proteins representative of all protein folds, to investigate the change in lysine-lysine distances resulting from native-state dynamics on the time-scale of tens of nanoseconds. We conclude that observed cross-links are consistent with a protein structure if the distance between cross-linked lysine Nζ atoms is less than the cross-linker length plus 11.3 Å. For DSS or BS3, this corresponds to a Cα to Cα distance of 30.4 Å. This analysis provides a theoretical basis for the widespread practice of adding a tolerance to the crosslinker length when comparing XL-MS results to structures, and indicates the appropriate values of an XLMS derived distance constraint to use in structural modeling.

  6. Extracellular surface residues of the α1B-adrenoceptor critical for G protein-coupled receptor function.

    PubMed

    Ragnarsson, Lotten; Andersson, Åsa; Thomas, Walter G; Lewis, Richard J

    2015-01-01

    Ligand binding and conformational changes that accompany signaling from G protein-coupled receptors (GPCRs) have mostly focused on the role of transmembrane helices and intracellular loop regions. However, recent studies, including several GPCRs cocrystallized with bound ligands, suggest that the extracellular surface (ECS) of GPCRs plays an important role in ligand recognition, selectivity, and binding, as well as potentially contributing to receptor activation and signaling. This study applied alanine-scanning mutagenesis to investigate the role of the complete ECS of the α1B-adrenoreceptor on norepinephrine (NE) potency, affinity, and efficacy. Half (24 of 48) of the ECS mutations significantly decreased NE potency in an inositol 1-phosphate assay. Most mutations reduced NE affinity (17) determined from [(3)H]prazosin displacement studies, whereas four mutations at the entrance to the NE binding pocket enhanced NE affinity. Removing the influence of NE affinity and receptor expression levels on NE potency gave a measure of NE efficacy, which was significantly decreased for 11 of 48 ECS mutants. These different effects tended to cluster to different regions of the ECS, which is consistent with different regions of the ECS playing discrete functional roles. Exposed ECS residues at the entrance to the NE binding pocket mostly affected NE affinity, whereas buried or structurally significant residues mostly affected NE efficacy. The broad potential for ECS mutations to affect GPCR function has relevance for the increasing number of nonsynonymous single nucleotide polymorphisms now being identified in GPCRs. PMID:25352041

  7. Mechanism of adenylate kinase. Are the essential lysines essential?

    PubMed

    Tian, G C; Yan, H G; Jiang, R T; Kishi, F; Nakazawa, A; Tsai, M D

    1990-05-01

    Using site-specific mutagenesis, we have probed the structural and functional roles of lysine-21 and lysine-27 of adenylate kinase (AK) from chicken muscle expressed in Escherichia coli. The two residues were chosen since according to the nuclear magnetic resonance (NMR) model [Mildvan, A. S., & Fry, D. C. (1987) Adv. Enzymol. 58, 241-313], they are located near the alpha- and the gamma-phosphates, respectively, of adenosine 5'-triphosphate (ATP) in the AK-MgATP complex. In addition, a lysine residue (Lys-21 in the case of AK) along with a glycine-rich loop is considered "essential" in the catalysis of kinases and other nucleotide binding proteins. The Lys-27 to methionine (K27M) mutant showed only slight increases in kcat and Km, but a substantial increase (1.8 kcal/mol) in the free energy of unfolding, relative to the WT AK. For proper interpretation of the steady-state kinetic data, viscosity-dependent kinetics was used to show that the chemical step is partially rate-limiting in the catalysis of AK. Computer modeling suggested that the folded form of K27M could gain stability (relative to the wild type) via hydrophobic interactions of Met-27 with Val-179 and Phe-183 and/or formation of a charge-transfer complex between Met-27 and Phe-183. The latter was supported by an upfield shift of the methyl protons of Met-27 in 1H NMR. Other than this, the 1H NMR spectrum of K27M is very similar to that of WT, suggesting little perturbation in the global or even local conformations.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2161682

  8. Targeting histone lysine demethylases — Progress, challenges, and the future☆

    PubMed Central

    Thinnes, Cyrille C.; England, Katherine S.; Kawamura, Akane; Chowdhury, Rasheduzzaman; Schofield, Christopher J.; Hopkinson, Richard J.

    2014-01-01

    N-Methylation of lysine and arginine residues has emerged as a major mechanism of transcriptional regulation in eukaryotes. In humans, Nε-methyllysine residue demethylation is catalysed by two distinct subfamilies of demethylases (KDMs), the flavin-dependent KDM1 subfamily and the 2-oxoglutarate- (2OG) dependent JmjC subfamily, which both employ oxidative mechanisms. Modulation of histone methylation status is proposed to be important in epigenetic regulation and has substantial medicinal potential for the treatment of diseases including cancer and genetic disorders. This article provides an introduction to the enzymology of the KDMs and the therapeutic possibilities and challenges associated with targeting them, followed by a review of reported KDM inhibitors and their mechanisms of action from kinetic and structural perspectives. This article is part of a Special Issue entitled: Methylation: A Multifaceted Modification — looking at transcription and beyond. PMID:24859458

  9. Acetylome analysis reveals the involvement of lysine acetylation in biosynthesis of antibiotics in Bacillus amyloliquefaciens

    PubMed Central

    Liu, Lin; Wang, Guangyuan; Song, Limin; Lv, Binna; Liang, Wenxing

    2016-01-01

    Lysine acetylation is a major post-translational modification that plays an important regulatory role in almost every aspects in both eukaryotes and prokaryotes. Bacillus amyloliquefaciens, a Gram-positive bacterium, is very effective for the control of plant pathogens. However, very little is known about the function of lysine acetylation in this organism. Here, we conducted the first lysine acetylome in B. amyloliquefaciens through a combination of highly sensitive immune-affinity purification and high-resolution LC−MS/MS. Overall, we identified 3268 lysine acetylation sites in 1254 proteins, which account for 32.9% of the total proteins in this bacterium. Till date, this is the highest ratio of acetylated proteins that have been identified in bacteria. Acetylated proteins are associated with a variety of biological processes and a large fraction of these proteins are involved in metabolism. Interestingly, for the first time, we found that about 71.1% (27/38) and 78.6% (22/28) of all the proteins tightly related to the synthesis of three types of pepketides and five families of lipopeptides were acetylated, respectively. These findings suggest that lysine acetylation plays a critical role in the regulation of antibiotics biosynthesis. These data serves as an important resource for further elucidation of the physiological role of lysine acetylation in B. amyloliquefaciens. PMID:26822828

  10. Linkages in thermal copolymers of lysine

    NASA Technical Reports Server (NTRS)

    Fox, S. W.; Suzuki, F.

    1975-01-01

    The thermal copolymerization of lysine with other alpha-amino acids was studied. The identity of the second amino acid influences various properties of the polymer obtained, including the proportion of alpha and epsilon linkages of lysine. A review of linkages in proteinoids indicates alpha and beta linkages for aspartic acid, alpha and gamma linkages for glutamic acid, alpha and epsilon linkages for lysine, and alpha linkages for other amino acids. Thermal proteinoids are thus more complex in types of linkage than are proteins.

  11. Linkages in thermal copolymers of lysine

    NASA Technical Reports Server (NTRS)

    Fox, S. W.; Suzuki, F.

    1976-01-01

    The thermal copolymerization of lysine with other alpha-amino acids has been studied further. The identity of the second amino acid influences various properties of the polymer obtained, including the proportion of alpha and epsilon linkages of lysine. A review of linkages in proteinoids indicates alpha and beta linkages for aspartic acid, alpha and gamma linkages for glutamic acid, alpha and epsilon linkages for lysine, and alpha linkages for other amino acids. Thermal proteinoids are thus more complex in types of linkage than are proteins

  12. Nitrate isotopes unveil distinct seasonal N-sources and the critical role of crop residues in groundwater contamination

    NASA Astrophysics Data System (ADS)

    Savard, Martine M.; Somers, George; Smirnoff, Anna; Paradis, Daniel; van Bochove, Eric; Liao, Shawna

    2010-02-01

    SummaryGlobally, fertilizers are identified as principle sources of nitrate in waters of intensely cultivated areas. Here this general concept is appraised on a seasonal basis over a two year period, under temperate climatic conditions. Water ( δ2H and δ18O) and nitrate ( δ15N and δ18O) isotopes in surface water and groundwater suggest that freshwater is acting as a transport vector conducting nitrate from agricultural soils to groundwater and ultimately to surface water. Measured nitrate isotopes of organic and inorganic fertilizers and of nitrate in groundwater are used to constrain a conceptual apportionment model quantifying the relative seasonal N contributions in an area of intense potato production. Source inputs differ strongly between the growing (summer and fall) and non-growing (winter and spring) periods. Chemical fertilizers and soil organic matter equally dominate and contribute to the growing period load, whereas soil organic matter dominates the non-growing period load, and accounts for over half of the overall annual nitrogen charge. These findings reveal the magnitude of nitrogen cycling by soil organic matter, and point to the benefits of controlling the timing of its nitrate release from this organic material. We conclude that strategies to attenuate contamination by nitrate in waters of temperate climate row-cropping regions must consider nitrogen cycling by soil organic matter, including the crucial role of crop residues throughout both the growing and non-growing seasons.

  13. Plastidic phosphoglycerate kinase from Phaeodactylum tricornutum: on the critical role of cysteine residues for the enzyme function.

    PubMed

    Bosco, María Belén; Aleanzi, Mabel Cristina; Iglesias, Alberto Álvaro

    2012-03-01

    Chloroplastidic phosphoglycerate kinase (PGKase) plays a key role in photosynthetic organisms, catalyzing a key step in the Calvin cycle. We performed the molecular cloning of the gene encoding chloroplastidic PGKase-1 in the diatom Phaeodactylum tricornutum. The recombinant enzyme was expressed in Escherichia coli, purified and characterized. Afterward, it showed similar kinetic properties than the enzyme studied from other organisms, although the diatom enzyme displayed distinctive responses to sulfhydryl reagents. The activity of the enzyme was found to be dependent on the redox status in the environment, determined by different compounds, including some of physiological function. Treatment with oxidant agents, such as diamide, hydrogen peroxide, glutathione and sodium nitroprusside resulted in enzyme inhibition. Recovery of activity was possible by subsequent incubation with reducing reagents such as dithiothreitol and thioredoxins (from E. coli and P. tricornutum). We determined two midpoint potentials of different regulatory redox centers, both values indicating that PGKase-1 might be sensitive to changes in the intracellular redox environment. The role of all the six Cys residues found in the diatom enzyme was analyzed by molecular modeling and site-directed mutagenesis. Results suggest key regulatory properties for P. tricornutum PGKase-1, which could be relevant for the functioning of photosynthetic carbon metabolism in diatoms. PMID:21816671

  14. Meropenem Population Pharmacokinetics in Critically Ill Patients with Septic Shock and Continuous Renal Replacement Therapy: Influence of Residual Diuresis on Dose Requirements

    PubMed Central

    Llaurado-Serra, Mireia; Vaquer, Sergi; Castro, Pedro; Rodríguez, Alejandro H.; Pontes, Caridad; Calvo, Gonzalo; Torres, Antoni; Martín-Loeches, Ignacio

    2015-01-01

    Meropenem dosing in critically ill patients with septic shock and continuous renal replacement therapy (CRRT) is complex, with the recommended maintenance doses being 500 mg to 1,000 mg every 8 h (q8h) to every 12 h. This multicenter study aimed to describe the pharmacokinetics (PKs) of meropenem in this population to identify the sources of PK variability and to evaluate different dosing regimens to develop recommendations based on clinical parameters. Thirty patients with septic shock and CRRT receiving meropenem were enrolled (153 plasma samples were tested). A population PK model was developed with data from 24 patients and subsequently validated with data from 6 patients using NONMEM software (v.7.3). The final model was characterized by CL = 3.68 + 0.22 · (residual diuresis/100) and V = 33.00 · (weight/73)2.07, where CL is total body clearance (in liters per hour), residual diuresis is the volume of residual diuresis (in milliliters per 24 h), and V is the apparent volume of distribution (in liters). CRRT intensity was not identified to be a CL modifier. Monte Carlo simulations showed that to maintain concentrations of the unbound fraction (fu) of drug above the MIC of the bacteria for 40% of dosing interval T (referred to as 40% of the ƒuT>MIC), a meropenem dose of 500 mg q8h as a bolus over 30 min would be sufficient regardless of the residual diuresis. If 100% of the ƒuT>MIC was chosen as the target, oligoanuric patients would require 500 mg q8h as a bolus over 30 min for the treatment of susceptible bacteria (MIC < 2 mg/liter), while patients with preserved diuresis would require the same dose given as an infusion over 3 h. If bacteria with MICs close to the resistance breakpoint (2 to 4 mg/liter) were to be treated with meropenem, a dose of 500 mg every 6 h would be necessary: a bolus over 30 min for oligoanuric patients and an infusion over 3 h for patients with preserved diuresis. Our results suggest that residual diuresis may be an easy and

  15. Miscanthus Establishment and Overwintering in the Midwest USA: A Regional Modeling Study of Crop Residue Management on Critical Minimum Soil Temperatures

    PubMed Central

    Kucharik, Christopher J.; VanLoocke, Andy; Lenters, John D.; Motew, Melissa M.

    2013-01-01

    Miscanthus is an intriguing cellulosic bioenergy feedstock because its aboveground productivity is high for low amounts of agrochemical inputs, but soil temperatures below −3.5°C could threaten successful cultivation in temperate regions. We used a combination of observed soil temperatures and the Agro-IBIS model to investigate how strategic residue management could reduce the risk of rhizome threatening soil temperatures. This objective was addressed using a historical (1978–2007) reconstruction of extreme minimum 10 cm soil temperatures experienced across the Midwest US and model sensitivity studies that quantified the impact of crop residue on soil temperatures. At observation sites and for simulations that had bare soil, two critical soil temperature thresholds (50% rhizome winterkill at −3.5°C and −6.0°C for different Miscanthus genotypes) were reached at rhizome planting depth (10 cm) over large geographic areas. The coldest average annual extreme 10 cm soil temperatures were between −8°C to −11°C across North Dakota, South Dakota, and Minnesota. Large portions of the region experienced 10 cm soil temperatures below −3.5°C in 75% or greater for all years, and portions of North and South Dakota, Minnesota, and Wisconsin experienced soil temperatures below −6.0°C in 50–60% of all years. For simulated management options that established varied thicknesses (1–5 cm) of miscanthus straw following harvest, extreme minimum soil temperatures increased by 2.5°C to 6°C compared to bare soil, with the greatest warming associated with thicker residue layers. While the likelihood of 10 cm soil temperatures reaching −3.5°C was greatly reduced with 2–5 cm of surface residue, portions of the Dakotas, Nebraska, Minnesota, and Wisconsin still experienced temperatures colder than −3.5°C in 50–80% of all years. Nonetheless, strategic residue management could help increase the likelihood of overwintering of miscanthus rhizomes in the first

  16. Negative Regulation of Interferon-induced Transmembrane Protein 3 by SET7-mediated Lysine Monomethylation*

    PubMed Central

    Shan, Zhao; Han, Qinglin; Nie, Jia; Cao, Xuezhi; Chen, Zuojia; Yin, Shuying; Gao, Yayi; Lin, Fang; Zhou, Xiaohui; Xu, Ke; Fan, Huimin; Qian, Zhikang; Sun, Bing; Zhong, Jin; Li, Bin; Tsun, Andy

    2013-01-01

    Although lysine methylation is classically known to regulate histone function, its role in modulating antiviral restriction factor activity remains uncharacterized. Interferon-induced transmembrane protein 3 (IFITM3) was found monomethylated on its lysine 88 residue (IFITM3-K88me1) to reduce its antiviral activity, mediated by the lysine methyltransferase SET7. Vesicular stomatitis virus and influenza A virus infection increased IFITM3-K88me1 levels by promoting the interaction between IFITM3 and SET7, suggesting that this pathway could be hijacked to support infection; conversely, IFN-α reduced IFITM3-K88me1 levels. These findings may have important implications in the design of therapeutics targeting protein methylation against infectious diseases. PMID:24129573

  17. Exploring the allosteric mechanism of dihydrodipicolinate synthase by reverse engineering of the allosteric inhibitor binding sites and its application for lysine production.

    PubMed

    Geng, Feng; Chen, Zhen; Zheng, Ping; Sun, Jibin; Zeng, An-Ping

    2013-03-01

    Dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) catalyzes the first committed reaction of L-lysine biosynthesis in bacteria and plants and is allosterically regulated by L-lysine. In previous studies, DHDPSs from different species were proved to have different sensitivity to L-lysine inhibition. In this study, we investigated the key determinants of feedback regulation between two industrially important DHDPSs, the L-lysine-sensitive DHDPS from Escherichia coli and L-lysine-insensitive DHDPS from Corynebacterium glutamicum, by sequence and structure comparisons and site-directed mutation. Feedback inhibition of E. coli DHDPS was successfully alleviated after substitution of the residues around the inhibitor's binding sites with those of C. glutamicum DHDPS. Interestingly, mutagenesis of the lysine binding sites of C. glutamicum DHDPS according to E. coli DHDPS did not recover the expected feedback inhibition but an activation of DHDPS by L-lysine, probably due to differences in the allosteic signal transduction in the DHDPS of these two organisms. Overexpression of L-lysine-insensitive E. coli DHDPS mutants in E. coli MG1655 resulted in an improvement of L-lysine production yield by 46 %. PMID:22644522

  18. SPOTing Acetyl-Lysine Dependent Interactions

    PubMed Central

    Picaud, Sarah; Filippakopoulos, Panagis

    2015-01-01

    Post translational modifications have been recognized as chemical signals that create docking sites for evolutionary conserved effector modules, allowing for signal integration within large networks of interactions. Lysine acetylation in particular has attracted attention as a regulatory modification, affecting chromatin structure and linking to transcriptional activation. Advances in peptide array technologies have facilitated the study of acetyl-lysine-containing linear motifs interacting with the evolutionary conserved bromodomain module, which specifically recognizes and binds to acetylated sequences in histones and other proteins. Here we summarize recent work employing SPOT peptide technology to identify acetyl-lysine dependent interactions and document the protocols adapted in our lab, as well as our efforts to characterize such bromodomain-histone interactions. Our results highlight the versatility of SPOT methods and establish an affordable tool for rapid access to potential protein/modified-peptide interactions involving lysine acetylation.

  19. Lysine-specific histone demethylases in normal and malignant hematopoiesis.

    PubMed

    Andricovich, Jaclyn; Kai, Yan; Tzatsos, Alexandros

    2016-09-01

    The epigenetic control of gene expression is central to the development of the hematopoietic system and the execution of lineage-specific transcriptional programs. During the last 10 years, mounting evidence has implicated the family of lysine-specific histone demethylases as critical regulators of normal hematopoiesis, whereas their deregulation is found in a broad spectrum of hematopoietic malignancies. Here, we review recent findings on the role of these enzymes in normal and malignant hematopoiesis and highlight how aberrant epigenetic regulation facilitates hematopoietic cell transformation through subversion of cell fate and lineage commitment programs. PMID:27208808

  20. A Method to determine lysine acetylation stoichiometries

    SciTech Connect

    Nakayasu, Ernesto S.; Wu, Si; Sydor, Michael A.; Shukla, Anil K.; Weitz, Karl K.; Moore, Ronald J.; Hixson, Kim K.; Kim, Jong Seo; Petyuk, Vladislav A.; Monroe, Matthew E.; Pasa-Tolic, Ljiljana; Qian, Weijun; Smith, Richard D.; Adkins, Joshua N.; Ansong, Charles

    2014-07-21

    A major bottleneck to fully understanding the functional aspects of lysine acetylation is the lack of stoichiometry information. Here we describe a mass spectrometry method using a combination of isotope labeling and detection of a diagnostic fragment ion to determine the stoichiometry of lysine acetylation on proteins globally. Using this technique, we determined the modification occupancy on hundreds of acetylated peptides from cell lysates and cross-validated the measurements via immunoblotting.

  1. The Conserved Lys-95 Charged Residue Cluster Is Critical for the Homodimerization and Enzyme Activity of Human Ribonucleotide Reductase Small Subunit M2*

    PubMed Central

    Chen, Xinhuan; Xu, Zhijian; Zhang, Lingna; Liu, Hongchuan; Liu, Xia; Lou, Meng; Zhu, Lijun; Huang, Bingding; Yang, Cai-Guang; Zhu, Weiliang; Shao, Jimin

    2014-01-01

    Ribonucleotide reductase (RR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides for DNA synthesis. Human RR small subunit M2 exists in a homodimer form. However, the importance of the dimer form to the enzyme and the related mechanism remain unclear. In this study, we tried to identify the interfacial residues that may mediate the assembly of M2 homodimer by computational alanine scanning based on the x-ray crystal structure. Co-immunoprecipitation, size exclusion chromatography, and RR activity assays showed that the K95E mutation in M2 resulted in dimer disassembly and enzyme activity inhibition. In comparison, the charge-exchanging double mutation of K95E and E98K recovered the dimerization and activity. Structural comparisons suggested that a conserved cluster of charged residues, including Lys-95, Glu-98, Glu-105, and Glu-174, at the interface may function as an ionic lock for M2 homodimer. Although the measurements of the radical and iron contents showed that the monomer (the K95E mutant) was capable of generating the diiron and tyrosyl radical cofactor, co-immunoprecipitation and competitive enzyme inhibition assays indicated that the disassembly of M2 dimer reduced its interaction with the large subunit M1. In addition, the immunofluorescent and fusion protein-fluorescent imaging analyses showed that the dissociation of M2 dimer altered its subcellular localization. Finally, the transfection of the wild-type M2 but not the K95E mutant rescued the G1/S phase cell cycle arrest and cell growth inhibition caused by the siRNA knockdown of M2. Thus, the conserved Lys-95 charged residue cluster is critical for human RR M2 homodimerization, which is indispensable to constitute an active holoenzyme and function in cells. PMID:24253041

  2. Mutation of critical serine residues in HIV-1 matrix result in an envelope incorporation defect which can be rescued by truncation of the gp41 cytoplasmic tail

    SciTech Connect

    Bhatia, Ajay K.; Kaushik, Rajnish; Campbell, Nancy A.; Pontow, Suzanne E.; Ratner, Lee

    2009-02-05

    The human immunodeficiency virus type 1 (HIV-1) matrix (MA) domain is involved in both early and late events of the viral life cycle. Simultaneous mutation of critical serine residues in MA has been shown previously to dramatically reduce phosphorylation of MA. However, the role of phosphorylation in viral replication remains unclear. Viruses harboring serine to alanine substitutions at positions 9, 67, 72, and 77 are severely impaired in their ability to infect target cells. In addition, the serine mutant viruses are defective in their ability to fuse with target cell membranes. Interestingly, both the fusion defect and the infectivity defect can be rescued by truncation of the long cytoplasmic tail of gp41 envelope protein (gp41CT). Sucrose density gradient analysis also reveals that these mutant viruses have reduced levels of gp120 envelope protein incorporated into the virions as compared to wild type virus. Truncation of the gp41CT rescues the envelope incorporation defect. Here we propose a model in which mutation of specific serine residues prevents MA interaction with lipid rafts during HIV-1 assembly and thereby impairs recruitment of envelope to the sites of viral budding.

  3. Lysine Activation and Functional Analysis of E2-Mediated Conjugation in the SUMO Pathway

    SciTech Connect

    Yunus,A.; Lima, C.

    2006-01-01

    E2 conjugating proteins that transfer ubiquitin and ubiquitin-like modifiers to substrate lysine residues must first activate the lysine nucleophile for conjugation. Genetic complementation revealed three side chains of the E2 Ubc9 that were crucial for normal growth. Kinetic analysis revealed modest binding defects but substantially lowered catalytic rates for these mutant alleles with respect to wild-type Ubc9. X-ray structures for wild-type and mutant human Ubc9-RanGAP1 complexes showed partial loss of contacts to the substrate lysine in mutant complexes. Computational analysis predicted pK perturbations for the substrate lysine, and Ubc9 mutations weakened pK suppression through improper side chain coordination. Biochemical studies with p53, RanGAP1 and the Nup358/RanBP2 E3 were used to determine rate constants and pK values, confirming both structural and computational predictions. It seems that Ubc9 uses an indirect mechanism to activate lysine for conjugation that may be conserved among E2 family members.

  4. Antibacterial Activity of a Novel Peptide-Modified Lysin Against Acinetobacter baumannii and Pseudomonas aeruginosa

    PubMed Central

    Yang, Hang; Wang, Mengyue; Yu, Junping; Wei, Hongping

    2015-01-01

    The global emergence of multidrug-resistant (MDR) bacteria is a growing threat to public health worldwide. Natural bacteriophage lysins are promising alternatives in the treatment of infections caused by Gram-positive pathogens, but not Gram-negative ones, like Acinetobacter baumannii and Pseudomonas aeruginosa, due to the barriers posed by their outer membranes. Recently, modifying a natural lysin with an antimicrobial peptide was found able to break the barriers, and to kill Gram-negative pathogens. Herein, a new peptide-modified lysin (PlyA) was constructed by fusing the cecropin A peptide residues 1–8 (KWKLFKKI) with the OBPgp279 lysin and its antibacterial activity was studied. PlyA showed good and broad antibacterial activities against logarithmic phase A. baumannii and P. aeruginosa, but much reduced activities against the cells in stationary phase. Addition of outer membrane permeabilizers (EDTA and citric acid) could enhance the antibacterial activity of PlyA against stationary phase cells. Finally, no antibacterial activity of PlyA could be observed in some bio-matrices, such as culture media, milk, and sera. In conclusion, we reported here a novel peptide-modified lysin with significant antibacterial activity against both logarithmic (without OMPs) and stationary phase (with OMPs) A. baumannii and P. aeruginosa cells in buffer, but further optimization is needed to achieve broad activity in diverse bio-matrices. PMID:26733995

  5. Identification of Lethal Mutations in Yeast Threonyl-tRNA Synthetase Revealing Critical Residues in Its Human Homolog*

    PubMed Central

    Ruan, Zhi-Rong; Fang, Zhi-Peng; Ye, Qing; Lei, Hui-Yan; Eriani, Gilbert; Zhou, Xiao-Long; Wang, En-Duo

    2015-01-01

    Aminoacyl-tRNA synthetases (aaRSs) are a group of ancient enzymes catalyzing aminoacylation and editing reactions for protein biosynthesis. Increasing evidence suggests that these critical enzymes are often associated with mammalian disorders. Therefore, complete determination of the enzymes functions is essential for informed diagnosis and treatment. Here, we show that a yeast knock-out strain for the threonyl-tRNA synthetase (ThrRS) gene is an excellent platform for such an investigation. Saccharomyces cerevisiae ThrRS has a unique modular structure containing four structural domains and a eukaryote-specific N-terminal extension. Using randomly mutated libraries of the ThrRS gene (thrS) and a genetic screen, a set of loss-of-function mutants were identified. The mutations affected the synthetic and editing activities and influenced the dimer interface. The results also highlighted the role of the N-terminal extension for enzymatic activity and protein stability. To gain insights into the pathological mechanisms induced by mutated aaRSs, we systematically introduced the loss-of-function mutations into the human cytoplasmic ThrRS gene. All mutations induced similar detrimental effects, showing that the yeast model could be used to study pathology-associated point mutations in mammalian aaRSs. PMID:25416776

  6. Identification of lethal mutations in yeast threonyl-tRNA synthetase revealing critical residues in its human homolog.

    PubMed

    Ruan, Zhi-Rong; Fang, Zhi-Peng; Ye, Qing; Lei, Hui-Yan; Eriani, Gilbert; Zhou, Xiao-Long; Wang, En-Duo

    2015-01-16

    Aminoacyl-tRNA synthetases (aaRSs) are a group of ancient enzymes catalyzing aminoacylation and editing reactions for protein biosynthesis. Increasing evidence suggests that these critical enzymes are often associated with mammalian disorders. Therefore, complete determination of the enzymes functions is essential for informed diagnosis and treatment. Here, we show that a yeast knock-out strain for the threonyl-tRNA synthetase (ThrRS) gene is an excellent platform for such an investigation. Saccharomyces cerevisiae ThrRS has a unique modular structure containing four structural domains and a eukaryote-specific N-terminal extension. Using randomly mutated libraries of the ThrRS gene (thrS) and a genetic screen, a set of loss-of-function mutants were identified. The mutations affected the synthetic and editing activities and influenced the dimer interface. The results also highlighted the role of the N-terminal extension for enzymatic activity and protein stability. To gain insights into the pathological mechanisms induced by mutated aaRSs, we systematically introduced the loss-of-function mutations into the human cytoplasmic ThrRS gene. All mutations induced similar detrimental effects, showing that the yeast model could be used to study pathology-associated point mutations in mammalian aaRSs. PMID:25416776

  7. Proton Affinity of Isomeric Dipeptides Containing Lysine and Non-Proteinogenic Lysine Homologues.

    PubMed

    Batoon, Patrick; Ren, Jianhua

    2016-08-18

    Conformational effects on the proton affinity of oligopeptides have been studied using six alanine (A)-based acetylated dipeptides containing a basic probe that is placed closest to either the C- or the N-terminus. The basic probe includes Lysine (Lys) and two nonproteinogenic Lys-homologues, ornithine (Orn) and 2,3-diaminopropionic acid (Dap). The proton affinities of the peptides have been determined using the extended Cooks kinetic method in a triple quadrupole mass spectrometer. Computational studies have been carried out to search for the lowest energy conformers and to calculate theoretical proton affinities as well as various molecular properties using the density functional theory. The dipeptides containing a C-terminal probe, ALys, AOrn, and ADap, were determined to have a higher proton affinity by 1-4 kcal/mol than the corresponding dipeptides containing an N-terminal probe, LysA, OrnA, and DapA. For either the C-probe peptides or the N-probe peptides, the proton affinity reduces systematically as the side-chain of the probe residue is shortened. The difference in the proton affinities between isomeric peptides is largely associated with the variation of the conformations. The peptides with higher values of the proton affinity adopt a relatively compact conformation such that the protonated peptides can be stabilized through more efficient internal solvation. PMID:27459294

  8. Structure of the lysine specific protease Kgp from Porphyromonas gingivalis, a target for improved oral health.

    PubMed

    Gorman, Michael A; Seers, Christine A; Michell, Belinda J; Feil, Susanne C; Huq, N Laila; Cross, Keith J; Reynolds, Eric C; Parker, Michael W

    2015-01-01

    The oral pathogen Porphyromonas gingivalis is a keystone pathogen in the development of chronic periodontitis. Gingipains, the principle virulence factors of P. gingivalis are multidomain, cell-surface proteins containing a cysteine protease domain. The lysine specific gingipain, Kgp, is a critical virulence factor of P. gingivalis. We have determined the X-ray crystal structure of the lysine-specific protease domain of Kgp to 1.6 Å resolution. The structure provides insights into the mechanism of substrate specificity and catalysis. PMID:25327141

  9. Structure of the lysine specific protease Kgp from Porphyromonas gingivalis, a target for improved oral health

    PubMed Central

    Gorman, Michael A; Seers, Christine A; Michell, Belinda J; Feil, Susanne C; Huq, N Laila; Cross, Keith J; Reynolds, Eric C; Parker, Michael W

    2015-01-01

    The oral pathogen Porphyromonas gingivalis is a keystone pathogen in the development of chronic periodontitis. Gingipains, the principle virulence factors of P. gingivalis are multidomain, cell-surface proteins containing a cysteine protease domain. The lysine specific gingipain, Kgp, is a critical virulence factor of P. gingivalis. We have determined the X-ray crystal structure of the lysine-specific protease domain of Kgp to 1.6 Å resolution. The structure provides insights into the mechanism of substrate specificity and catalysis. PMID:25327141

  10. Mapping Key Residues of ISD11 Critical for NFS1-ISD11 Subcomplex Stability: IMPLICATIONS IN THE DEVELOPMENT OF MITOCHONDRIAL DISORDER, COXPD19.

    PubMed

    Saha, Prasenjit Prasad; Srivastava, Shubhi; Kumar S K, Praveen; Sinha, Devanjan; D'Silva, Patrick

    2015-10-23

    Biogenesis of the iron-sulfur (Fe-S) cluster is an indispensable process in living cells. In mammalian mitochondria, the initial step of the Fe-S cluster assembly process is assisted by the NFS1-ISD11 complex, which delivers sulfur to scaffold protein ISCU during Fe-S cluster synthesis. Although ISD11 is an essential protein, its cellular role in Fe-S cluster biogenesis is still not defined. Our study maps the important ISD11 amino acid residues belonging to putative helix 1 (Phe-40), helix 3 (Leu-63, Arg-68, Gln-69, Ile-72, Tyr-76), and C-terminal segment (Leu-81, Glu-84) are critical for in vivo Fe-S cluster biogenesis. Importantly, mutation of these conserved ISD11 residues into alanine leads to its compromised interaction with NFS1, resulting in reduced stability and enhanced aggregation of NFS1 in the mitochondria. Due to altered interaction with ISD11 mutants, the levels of NFS1 and Isu1 were significantly depleted, which affects Fe-S cluster biosynthesis, leading to reduced electron transport chain complex (ETC) activity and mitochondrial respiration. In humans, a clinically relevant ISD11 mutation (R68L) has been associated in the development of a mitochondrial genetic disorder, COXPD19. Our findings highlight that the ISD11 R68A/R68L mutation display reduced affinity to form a stable subcomplex with NFS1, and thereby fails to prevent NFS1 aggregation resulting in impairment of the Fe-S cluster biogenesis. The prime affected machinery is the ETC complex, which showed compromised redox properties, causing diminished mitochondrial respiration. Furthermore, the R68L ISD11 mutant displayed accumulation of mitochondrial iron and reactive oxygen species, leading to mitochondrial dysfunction, which correlates with the phenotype observed in COXPD19 patients. PMID:26342079

  11. A Combined Genetic-Proteomic Approach Identifies Residues within Dengue Virus NS4B Critical for Interaction with NS3 and Viral Replication

    PubMed Central

    Chatel-Chaix, Laurent; Fischl, Wolfgang; Scaturro, Pietro; Cortese, Mirko; Kallis, Stephanie; Bartenschlager, Marie; Fischer, Bernd

    2015-01-01

    ABSTRACT Dengue virus (DENV) infection causes the most prevalent arthropod-borne viral disease worldwide. Approved vaccines are not available, and targets suitable for the development of antiviral drugs are lacking. One possible drug target is nonstructural protein 4B (NS4B), because it is absolutely required for virus replication; however, its exact role in the DENV replication cycle is largely unknown. With the aim of mapping NS4B determinants critical for DENV replication, we performed a reverse genetic screening of 33 NS4B mutants in the context of an infectious DENV genome. While the majority of these mutations were lethal, for several of them, we were able to select for second-site pseudoreversions, most often residing in NS4B and restoring replication competence. To identify all viral NS4B interaction partners, we engineered a fully viable DENV genome encoding an affinity-tagged NS4B. Mass spectrometry-based analysis of the NS4B complex isolated from infected cells identified the NS3 protease/helicase as a major interaction partner of NS4B. By combining the genetic complementation map of NS4B with a replication-independent expression system, we identified the NS4B cytosolic loop—more precisely, amino acid residue Q134—as a critical determinant for NS4B-NS3 interaction. An alanine substitution at this site completely abrogated the interaction and DENV RNA replication, and both were restored by pseudoreversions A69S and A137V. This strict correlation between the degree of NS4B-NS3 interaction and DENV replication provides strong evidence that this viral protein complex plays a pivotal role during the DENV replication cycle, hence representing a promising target for novel antiviral strategies. IMPORTANCE With no approved therapy or vaccine against dengue virus infection, the viral nonstructural protein 4B (NS4B) represents a possible drug target, because it is indispensable for virus replication. However, little is known about its precise structure and

  12. The effect of glutamic acid side chain on acidity constant of lysine in beta-sheet: A density functional theory study

    NASA Astrophysics Data System (ADS)

    Sargolzaei, M.; Afshar, M.; Sadeghi, M. S.; Kavee, M.

    2014-07-01

    In this work, the possibility of proton transfer between side chain of lysine and glutamic acid in peptide of Glu--Ala-Lys+ was demonstrated using density functional theory (DFT). We have shown that the proton transfer takes place between side chain of glutamic and lysine residues through the hydrogen bond formation. The structures of transition state for proton transfer reaction were detected in gas and solution phases. Our kinetic studies show that the proton transfer reaction rate in gas phase is higher than solution phase. The ionization constant (p K a) value of lysine residue in peptide was estimated 1.039 which is lower than intrinsic p K a of lysine amino acid.

  13. NMR determination of lysine pKa values in the Pol lambda lyase domain: mechanistic implications.

    PubMed

    Gao, Guanghua; DeRose, Eugene F; Kirby, Thomas W; London, Robert E

    2006-02-14

    The base excision repair (BER) process requires removal of an abasic deoxyribose-5-phosphate group, a catalytic activity that has been demonstrated for the N-terminal 8 kDa domain of DNA polymerase beta (Pol beta), and for the homologous domain of DNA polymerase lambda (Pol lambda). Previous studies have demonstrated that this activity results from formation of a Schiff base adduct of the abasic deoxyribose C-1' with a lysine residue (K312 in the case of Pol lambda), followed by a beta-elimination reaction. To better understand the underlying chemistry, we have determined pKa values for the lysine residues in the Pol lambda lyase domain labeled with [epsilon-13C]lysine. At neutral pH, the H(epsilon) protons on 3 of the 10 lysine residues in this domain, K287, K291, and K312, exhibit chemical shift inequivalence that results from immobilization of the lysyl side chains. For K287 and K291, this results from the K287-E261 and K291-E298 salt bridge interactions, while for K312, immobilization apparently results from steric and hydrogen-bonding interactions that constrain the position of the lysine side chain. The pKa value of K312 is depressed to 9.58, a value indicating that at physiological pH K312 will exist predominantly in the protonated form. Titration of the domain with hairpin DNA containing a 5'-tetrahydrofuran terminus to model the abasic site produced shifts of the labeled lysine resonances that were in fast exchange but appeared to be complete at a stoichiometry of approximately 1:1.3, consistent with a dissociation constant of approximately 1 microM. The epsilon-proton shifts of K273 were the most sensitive to the addition of the DNA, apparently due to changes in the relative orientation between K273 and W274 in the DNA complex. The average pKa values increased by 0.55, consistent with the formation of some DNA-lysine salt bridges and with the general pH increase expected to result from a reduction in the net positive charge of the complex. A general

  14. Formation equilibria of nickel complexes with glycyl-histidyl-lysine and two synthetic analogues.

    PubMed

    Conato, Chiara; Kozłowski, Henryk; Swiatek-Kozłowska, Jolanta; Młynarz, Piotr; Remelli, Maurizio; Silvestri, Sergio

    2004-01-01

    Complex-formation equilibria between the Ni(II) ion and the natural tripeptide glycyl-L-histidyl-L-lysine have been investigated. Two synthetic analogues, where the histidine residue has been substituted with L-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid (L-Spinacine) and L-1,2,3,4-tetrahydro-isoquinolin-3-carboxylic acid (Tic), respectively, have been considered, as well. Different experimental techniques have been employed: potentiometry, calorimetry, visible spectrophotometry and CD spectroscopy. Structural hypotheses on the main complex species are suggested. Evidences on the formation of tetrameric species with the first ligand are shown. No involvement of the side-chain amino group of lysine residue in metal ion coordination was found. PMID:14659644

  15. Molecular Evolution of the Substrate Specificity of Chloroplastic Aldolases/Rubisco Lysine Methyltransferases in Plants.

    PubMed

    Ma, Sheng; Martin-Laffon, Jacqueline; Mininno, Morgane; Gigarel, Océane; Brugière, Sabine; Bastien, Olivier; Tardif, Marianne; Ravanel, Stéphane; Alban, Claude

    2016-04-01

    Rubisco and fructose-1,6-bisphosphate aldolases (FBAs) are involved in CO2 fixation in chloroplasts. Both enzymes are trimethylated at a specific lysine residue by the chloroplastic protein methyltransferase LSMT. Genes coding LSMT are present in all plant genomes but the methylation status of the substrates varies in a species-specific manner. For example, chloroplastic FBAs are naturally trimethylated in both Pisum sativum and Arabidopsis thaliana, whereas the Rubisco large subunit is trimethylated only in the former species. The in vivo methylation status of aldolases and Rubisco matches the catalytic properties of AtLSMT and PsLSMT, which are able to trimethylate FBAs or FBAs and Rubisco, respectively. Here, we created chimera and site-directed mutants of monofunctional AtLSMT and bifunctional PsLSMT to identify the molecular determinants responsible for substrate specificity. Our results indicate that the His-Ala/Pro-Trp triad located in the central part of LSMT enzymes is the key motif to confer the capacity to trimethylate Rubisco. Two of the critical residues are located on a surface loop outside the methyltransferase catalytic site. We observed a strict correlation between the presence of the triad motif and the in vivo methylation status of Rubisco. The distribution of the motif into a phylogenetic tree further suggests that the ancestral function of LSMT was FBA trimethylation. In a recent event during higher plant evolution, this function evolved in ancestors of Fabaceae, Cucurbitaceae, and Rosaceae to include Rubisco as an additional substrate to the archetypal enzyme. Our study provides insight into mechanisms by which SET-domain protein methyltransferases evolve new substrate specificity. PMID:26785049

  16. Insights into the Specificity of Lysine Acetyltransferases*

    PubMed Central

    Tucker, Alex C.; Taylor, Keenan C.; Rank, Katherine C.; Rayment, Ivan; Escalante-Semerena, Jorge C.

    2014-01-01

    Reversible lysine acetylation by protein acetyltransferases is a conserved regulatory mechanism that controls diverse cellular pathways. Gcn5-related N-acetyltransferases (GNATs), named after their founding member, are found in all domains of life. GNATs are known for their role as histone acetyltransferases, but non-histone bacterial protein acetytransferases have been identified. Only structures of GNAT complexes with short histone peptide substrates are available in databases. Given the biological importance of this modification and the abundance of lysine in polypeptides, how specificity is attained for larger protein substrates is central to understanding acetyl-lysine-regulated networks. Here we report the structure of a GNAT in complex with a globular protein substrate solved to 1.9 Å. GNAT binds the protein substrate with extensive surface interactions distinct from those reported for GNAT-peptide complexes. Our data reveal determinants needed for the recognition of a protein substrate and provide insight into the specificity of GNATs. PMID:25381442

  17. SET7/9 Catalytic Mutants Reveal the Role of Active Site Water Molecules in Lysine Multiple Methylation

    SciTech Connect

    Del Rizzo, Paul A.; Couture, Jean-François; Dirk, Lynnette M.A.; Strunk, Bethany S.; Roiko, Marijo S.; Brunzelle, Joseph S.; Houtz, Robert L.; Trievel, Raymond C.

    2010-11-15

    SET domain lysine methyltransferases (KMTs) methylate specific lysine residues in histone and non-histone substrates. These enzymes also display product specificity by catalyzing distinct degrees of methylation of the lysine {epsilon}-amino group. To elucidate the molecular mechanism underlying this specificity, we have characterized the Y245A and Y305F mutants of the human KMT SET7/9 (also known as KMT7) that alter its product specificity from a monomethyltransferase to a di- and a trimethyltransferase, respectively. Crystal structures of these mutants in complex with peptides bearing unmodified, mono-, di-, and trimethylated lysines illustrate the roles of active site water molecules in aligning the lysine {epsilon}-amino group for methyl transfer with S-adenosylmethionine. Displacement or dissociation of these solvent molecules enlarges the diameter of the active site, accommodating the increasing size of the methylated {epsilon}-amino group during successive methyl transfer reactions. Together, these results furnish new insights into the roles of active site water molecules in modulating lysine multiple methylation by SET domain KMTs and provide the first molecular snapshots of the mono-, di-, and trimethyl transfer reactions catalyzed by these enzymes.

  18. Defining the Orphan Functions of Lysine Acetyltransferases

    PubMed Central

    2016-01-01

    Long known for their role in histone acetylation, recent studies have demonstrated that lysine acetyltransferases also carry out distinct “orphan” functions. These activities impact a wide range of biological phenomena including metabolism, RNA modification, nuclear morphology, and mitochondrial function. Here, we review the discovery and characterization of orphan lysine acetyltransferase functions. In addition to highlighting the evidence and biological role for these functions in human disease, we discuss the part emerging chemical tools may play in investigating this versatile enzyme superfamily. PMID:25591746

  19. Interfacing protein lysine acetylation and protein phosphorylation

    PubMed Central

    Tran, Hue T.; Uhrig, R. Glen; Nimick, Mhairi; Moorhead, Greg B.

    2012-01-01

    Recognition that different protein covalent modifications can operate in concert to regulate a single protein has forced us to re-think the relationship between amino acid side chain modifications and protein function. Results presented by Tran et al. 2012 demonstrate the association of a protein phosphatase (PP2A) with a histone/lysine deacetylase (HDA14) on plant microtubules along with a histone/lysine acetyltransferase (ELP3). This finding reveals a regulatory interface between two prevalent covalent protein modifications, protein phosphorylation and acetylation, emphasizing the integrated complexity of post-translational protein regulation found in nature. PMID:22827947

  20. A Study on the Effect of Surface Lysine to Arginine Mutagenesis on Protein Stability and Structure Using Green Fluorescent Protein

    PubMed Central

    Sokalingam, Sriram; Raghunathan, Govindan; Soundrarajan, Nagasundarapandian; Lee, Sun-Gu

    2012-01-01

    Two positively charged basic amino acids, arginine and lysine, are mostly exposed to protein surface, and play important roles in protein stability by forming electrostatic interactions. In particular, the guanidinium group of arginine allows interactions in three possible directions, which enables arginine to form a larger number of electrostatic interactions compared to lysine. The higher pKa of the basic residue in arginine may also generate more stable ionic interactions than lysine. This paper reports an investigation whether the advantageous properties of arginine over lysine can be utilized to enhance protein stability. A variant of green fluorescent protein (GFP) was created by mutating the maximum possible number of lysine residues on the surface to arginines while retaining the activity. When the stability of the variant was examined under a range of denaturing conditions, the variant was relatively more stable compared to control GFP in the presence of chemical denaturants such as urea, alkaline pH and ionic detergents, but the thermal stability of the protein was not changed. The modeled structure of the variant indicated putative new salt bridges and hydrogen bond interactions that help improve the rigidity of the protein against different chemical denaturants. Structural analyses of the electrostatic interactions also confirmed that the geometric properties of the guanidinium group in arginine had such effects. On the other hand, the altered electrostatic interactions induced by the mutagenesis of surface lysines to arginines adversely affected protein folding, which decreased the productivity of the functional form of the variant. These results suggest that the surface lysine mutagenesis to arginines can be considered one of the parameters in protein stability engineering. PMID:22792305

  1. Antimicrobial activity of chicken NK-lysin against Eimeria sporozoites

    Technology Transfer Automated Retrieval System (TEKTRAN)

    NK-lysin is an antimicrobial and antitumor polypeptide that is considered to play an important role during innate immunity. Chicken NK-lysin is a member of the saposin-like protein family and exhibits potent antitumor cell activity. To evaluate the antimicrobial properties of chicken NK-lysin, we ex...

  2. Radioactive Lysine in Protein Metabolism Studies

    DOE R&D Accomplishments Database

    Miller, L. L.; Bale, W. F.; Yuile, C. L.; Masters, R. E.; Tishkoff, G. H.; Whipple,, G. H.

    1950-01-09

    Studies of incorporation of DL-lysine in various body proteins of the dog; the time course of labeled blood proteins; and apparent rate of disappearance of labeled plasma proteins for comparison of behavior of the plasma albumin and globulin fractions; shows more rapid turn over of globulin fraction.

  3. 21 CFR 582.5411 - Lysine.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Lysine. 582.5411 Section 582.5411 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  4. 21 CFR 582.5411 - Lysine.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Lysine. 582.5411 Section 582.5411 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  5. Weight gain, feed conversion efficiency and plasma free lysine as response criteria in evaluating supplements of lysine plus threonine and lysine plus tryptophan to deficient diets for rats.

    PubMed

    Frydrych, Z; Heger, J

    1986-08-01

    Two experiments were conducted on growing male SPF-rats to compare weight gain, feed conversion efficiency and plasma free lysine concentration as response criteria in evaluating adequacy of lysine plus threonine and lysine plus tryptophan supplements to the deficient diets. Two basal semisynthetic diets were prepared limiting in lysine and threonine (Expt. 1) and lysine and tryptophan (Expt. 2). The addition of graded supplements to the basal diets of L-lysine X HCl alone (0.2; 0.4; 0.6; 0.8 and 1.0% of diet) induced imbalance of amino acids resulting in low level of daily weight gain and feed conversion efficiency. Plasma free lysine concentration started to grow linearly from the first supplement of L-lysine X HCl. If rats were fed the diets containing identical supplements of L-lysine X HCl in combination with two supplements of L-threonine (0.2 and 0.4% of diet, Expt. 1) or L-tryptophan (0.05 and 0.1% of diet, Expt. 2), plasma free lysine started to increase before supplements of amino acids were adequate to support maximum weight gain and feed conversion efficiency. this difference in response seems to be caused by different feeding regiment during the growth period of the experiments (ad libitum) and training period prior to blood sampling (feeding twice daily). PMID:3098208

  6. Toxicity and critical body residues of Cd, Cu and Cr in the aquatic oligochaete Tubifex tubifex (Müller) based on lethal and sublethal effects.

    PubMed

    Méndez-Fernández, Leire; Martínez-Madrid, Maite; Rodriguez, Pilar

    2013-12-01

    The aim of the present study was to estimate critical body residues (CBRs) of three metals [cadmium (Cd), copper (Cu), chromium (Cr)] in the aquatic oligochaete Tubifex tubifex based on lethal (LBR) and sublethal effects (CBR), and to discuss the relevance of the exposure to sediment for deriving CBR. Toxicity parameters (LC50, EC50, LBR50 and CBR50) were estimated for each metal by means of data on survival and on several sublethal variables measured in short-term (4 days), water-only exposures and in long-term, chronic (14 and 28 days) exposures using metal-spiked sediment. Sublethal endpoints included autotomy in short-term exposure, as well as reproduction and growth in chronic bioassays. LBR50 and CBR50 were 3-6 times higher in sediment than in water-only exposure to Cd and about 2-11 times higher for Cu, depending on the measured endpoint; however, for Cr these parameters varied only by a factor of 1.2. Cu and Cr LBR50 and CBR50 values in 96 h water-only exposure were very similar (survival 2.39 μmol Cu g(-1) dw, 2.73 μmol Cr g(-1) dw; autotomy 0.53 μmol Cu g(-1) dw, 0.78 μmol Cr g(-1) dw). However, in metal-spiked sediments, 28 d CBR50 values for autotomy, reproduction and growth ranged 6.76-29.54 μmol g(-1) dw for Cd, 3.88-6.23 μmol g(-1) dw for Cu, 0.65 μmol g(-1) dw for Cr (calculated only on total number of young). Exposure conditions (time and presence/absence of sediment) seem to be influential in deriving metal CBR values of Cd and Cu, while appear to be irrelevant for Cr. Thus, CBR approach for metals is complex and tissue residue-toxicity relationship is not directly applicable so far. PMID:24085604

  7. Insights into Lysine Deacetylation of Natively Folded Substrate Proteins by Sirtuins.

    PubMed

    Knyphausen, Philipp; de Boor, Susanne; Kuhlmann, Nora; Scislowski, Lukas; Extra, Antje; Baldus, Linda; Schacherl, Magdalena; Baumann, Ulrich; Neundorf, Ines; Lammers, Michael

    2016-07-01

    Sirtuins are NAD(+)-dependent lysine deacylases, regulating a variety of cellular processes. The nuclear Sirt1, the cytosolic Sirt2, and the mitochondrial Sirt3 are robust deacetylases, whereas the other sirtuins have preferences for longer acyl chains. Most previous studies investigated sirtuin-catalyzed deacylation on peptide substrates only. We used the genetic code expansion concept to produce natively folded, site-specific, and lysine-acetylated Sirt1-3 substrate proteins, namely Ras-related nuclear, p53, PEPCK1, superoxide dismutase, cyclophilin D, and Hsp10, and analyzed the deacetylation reaction. Some acetylated proteins such as Ras-related nuclear, p53, and Hsp10 were robustly deacetylated by Sirt1-3. However, other reported sirtuin substrate proteins such as cyclophilin D, superoxide dismutase, and PEPCK1 were not deacetylated. Using a structural and functional approach, we describe the ability of Sirt1-3 to deacetylate two adjacent acetylated lysine residues. The dynamics of this process have implications for the lifetime of acetyl modifications on di-lysine acetylation sites and thus constitute a new mechanism for the regulation of proteins by acetylation. Our studies support that, besides the primary sequence context, the protein structure is a major determinant of sirtuin substrate specificity. PMID:27226597

  8. Engineering of Corynebacterium glutamicum with an NADPH-generating glycolytic pathway for L-lysine production.

    PubMed

    Takeno, Seiki; Murata, Ryosuke; Kobayashi, Ryosuke; Mitsuhashi, Satoshi; Ikeda, Masato

    2010-11-01

    A sufficient supply of NADPH is a critical factor in l-lysine production by Corynebacterium glutamicum. Endogenous NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) of C. glutamicum was replaced with nonphosphorylating NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (GapN) of Streptococcus mutans, which catalyzes the reaction of glyceraldehyde 3-phosphate to 3-phosphoglycerate with the reduction of NADP(+) to NADPH, resulting in the reconstruction of the functional glycolytic pathway. Although the growth of the engineered strain on glucose was significantly retarded, a suppressor mutant with an increased ability to utilize sugars was spontaneously isolated from the engineered strain. The suppressor mutant was characterized by the properties of GapN as well as the nucleotide sequence of the gene, confirming that no change occurred in either the activity or the basic properties of GapN. The suppressor mutant was engineered into an l-lysine-producing strain by plasmid-mediated expression of the desensitized lysC gene, and the performance of the mutant as an l-lysine producer was evaluated. The amounts of l-lysine produced by the suppressor mutant were larger than those produced by the reference strain (which was created by replacement of the preexisting gapN gene in the suppressor mutant with the original gapA gene) by ∼70% on glucose, ∼120% on fructose, and ∼100% on sucrose, indicating that the increased l-lysine production was attributed to GapN. These results demonstrate effective l-lysine production by C. glutamicum with an additional source of NADPH during glycolysis. PMID:20851994

  9. Characterization of Murine Cytomegalovirus m157 from Infected Cells and Identification of Critical Residues Mediating Recognition by the NK Cell Receptor, Ly49H

    PubMed Central

    Davis, Aja H.; Guseva, Natalya V.; Ball, Brianne L.; Heusel, Jonathan W.

    2008-01-01

    Activated natural killer (NK) cells mediate potent cytolytic and secretory effector functions, and are vital components of the early antiviral immune response. NK cell activities are regulated by the assortment of inhibitory receptors that recognize major histocompatibility class I ligands expressed on healthy cells and activating receptors that recognize inducible host ligands or ligands that are not well characterized. The activating Ly49H receptor of mouse NK cells is unique in that it specifically recognizes a virally encoded ligand, the m157 glycoprotein of murine cytomegalovirus (MCMV). The Ly49H-m157 interaction underlies a potent resistance mechanism (Cmv1) in C57BL/6 mice, and serves as an excellent model in which to understand how NK cells are specifically activated in vivo, as similar receptor systems are operative for human NK cells. For transduced cells expressing m157 in isolation and for MCMV-infected cells, we show that m157 is expressed in multiple isoforms with marked differences in abundance between infected fibroblasts (high) and macrophages (low). At the cell surface m157 is exclusively a glycosylphosphatidylinositol-associated protein in MCMV-infected cells. Through random and site-directed mutagenesis of m157 we identify unique residues that provide for efficient cell surface expression of m157, but fail to activate Ly49H-expressing reporter cells. These m157 mutations are predicted to alter the conformation of a putative m157 interface with Ly49H, one that relies on the position of a critical α0-helix of m157. These findings support an emerging model for a novel interaction between this important NK cell receptor and its viral ligand. PMID:18566392

  10. Critical Roles of Two Hydrophobic Residues within Human Glucose Transporter 9 (hSLC2A9) in Substrate Selectivity and Urate Transport*

    PubMed Central

    Long, Wentong; Panwar, Pankaj; Witkowska, Kate; Wong, Kenneth; O'Neill, Debbie; Chen, Xing-Zhen; Lemieux, M. Joanne; Cheeseman, Chris I.

    2015-01-01

    High blood urate levels (hyperuricemia) have been found to be a significant risk factor for cardiovascular diseases and inflammatory arthritis, such as hypertension and gout. Human glucose transporter 9 (hSLC2A9) is an essential protein that mainly regulates urate/hexose homeostasis in human kidney and liver. hSLC2A9 is a high affinity-low capacity hexose transporter and a high capacity urate transporter. Our previous studies identified a single hydrophobic residue in trans-membrane domain 7 of class II glucose transporters as a determinant of fructose transport. A mutation of isoleucine 335 to valine (I355V) in hSLC2A9 can reduce fructose transport while not affecting glucose fluxes. This current study demonstrates that the I335V mutant transports urate similarly to the wild type hSLC2A9; however, Ile-335 is necessary for urate/fructose trans-acceleration exchange to occur. Furthermore, Trp-110 is a critical site for urate transport. Two structural models of the class II glucose transporters, hSLC2A9 and hSLC2A5, based on the crystal structure of hSLC2A1 (GLUT1), reveal that Ile-335 (or the homologous Ile-296 in hSLC2A5) is a key component for protein conformational changes when the protein translocates substrates. The hSLC2A9 model also predicted that Trp-110 is a crucial site that could directly interact with urate during transport. Together, these studies confirm that hSLC2A9 transports both urate and fructose, but it interacts with them in different ways. Therefore, this study advances our understanding of how hSLC2A9 mediates urate and fructose transport, providing further information for developing pharmacological agents to treat hyperuricemia and related diseases, such as gout, hypertension, and diabetes. PMID:25922070

  11. QuEChERS sample preparation for the determination of pesticides and other organic residues in environmental matrices: a critical review.

    PubMed

    Bruzzoniti, Maria Concetta; Checchini, Leonardo; De Carlo, Rosa Maria; Orlandini, Serena; Rivoira, Luca; Del Bubba, Massimo

    2014-07-01

    Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) is an extraction and clean-up technique originally developed for recovering pesticide residues from fruits and vegetables. Since its introduction, and until December 2013, about 700 papers have been published using the QuEChERS technique, according to a literature overview carried out using SciFinder, Elsevier SciVerse, and Google search engines. Most of these papers were dedicated to pesticide multiresidue analysis in food matrices, and this topic has been thoroughly reviewed over recent years. The QuEChERS approach is now rapidly developing beyond its original field of application to analytes other than pesticides, and matrices other than food, such as biological fluids and non-edible plants, including Chinese medicinal plants. Recently, the QuEChERS concept has spread to environmental applications by analyzing not only pesticides but also other compounds of environmental concern in soil, sediments, and water. To the best of our knowledge, QuEChERS environmental applications have not been reviewed so far; therefore, in this contribution, after a general discussion on the evolution and changes of the original QuEChERS method, a critical survey of the literature regarding environmental applications of conventional and modified QuEChERS methodology is provided. The overall recoveries obtained with QuEChERS and other extraction approaches (e.g., accelerated solvent extraction, ultrasonic solvent extraction, liquid/solid extraction, and soxhlet extraction) were compared, providing evidence for QuEChERS higher recoveries for various classes of compounds, such as biopesticides, chloroalkanes, phenols, and perfluoroalkyl substances. The role of physicochemical properties of soil (i.e., clay and organic carbon content, as well as cation exchange capacity) and target analytes (i.e., log KOW, water solubility, and vapor pressure) were also evaluated in order to interpret recovery and matrix effect data. PMID

  12. Isoxazole-Derived Amino Acids are Bromodomain-Binding Acetyl-Lysine Mimics: Incorporation into Histone H4 Peptides and Histone H3.

    PubMed

    Sekirnik Née Measures, Angelina R; Hewings, David S; Theodoulou, Natalie H; Jursins, Lukass; Lewendon, Katie R; Jennings, Laura E; Rooney, Timothy P C; Heightman, Tom D; Conway, Stuart J

    2016-07-11

    A range of isoxazole-containing amino acids was synthesized that displaced acetyl-lysine-containing peptides from the BAZ2A, BRD4(1), and BRD9 bromodomains. Three of these amino acids were incorporated into a histone H4-mimicking peptide and their affinity for BRD4(1) was assessed. Affinities of the isoxazole-containing peptides are comparable to those of a hyperacetylated histone H4-mimicking cognate peptide, and demonstrated a dependence on the position at which the unnatural residue was incorporated. An isoxazole-based alkylating agent was developed to selectively alkylate cysteine residues in situ. Selective monoalkylation of a histone H4-mimicking peptide, containing a lysine to cysteine residue substitution (K12C), resulted in acetyl-lysine mimic incorporation, with high affinity for the BRD4 bromodomain. The same technology was used to alkylate a K18C mutant of histone H3. PMID:27264992

  13. The role of lysine 186 in HIV-1 integrase multimerization

    SciTech Connect

    Berthoux, Lionel; Sebastian, Sarah; Muesing, Mark A.; Luban, Jeremy . E-mail: luban@irb.unisi.ch

    2007-07-20

    HIV-1 integrase (IN) catalyzes biochemical reactions required for viral cDNA insertion into host cell chromosomal DNA, an essential step in the HIV-1 replication cycle. In one of these reactions, the two ends of the linear viral cDNA are believed to be simultaneously ligated to chromosomal DNA by a tetrameric form of IN. The structure of the full-length IN tetramer is not known but a model consisting of the N-terminal domain and the catalytic core revealed basic residues 186 to 188 at the interface between the two IN dimers. We found that alteration of these residues, in particular changing IN lysine residue 186 to glutamate (K186Q), impairs IN oligomerization in the yeast two-hybrid system and decreases oligomeric forms of IN within virions. When expressed independently of other viral proteins in human cells, IN-K186Q did not concentrate in the nucleus as did wild-type IN. Co-expression of wild-type IN restored the multimerization defects of IN-K186Q, in both the two-hybrid system and in virions, and also rescued the nuclear targeting defects. Virions bearing IN-K186Q were not infectious in a single cycle of replication but when mixed virions containing two different IN mutants were produced, IN-K186Q was capable of complementing the catalytically inactive mutant IN-D116A. Our biochemical and functional data support the crystallographic model in which IN residue K186 lies at the interface between IN dimers and suggest that tetramerization is important, not only for concerted integration, but also for IN nuclear targeting.

  14. Bacterial Lysine Decarboxylase Influences Human Dental Biofilm Lysine Content, Biofilm Accumulation and Sub-Clinical Gingival Inflammation

    PubMed Central

    Lohinai, Z.; Keremi, B.; Szoko, E.; Tabi, T.; Szabo, C.; Tulassay, Z.; Levine, M.

    2012-01-01

    Background Dental biofilms contain a protein that inhibits mammalian cell growth, possibly lysine decarboxylase from Eikenella corrodens. This enzyme decarboxylates lysine, an essential amino acid for dentally attached cell turnover in gingival sulci. Lysine depletion may stop this turnover, impairing the barrier to bacterial compounds. The aims of this study were to determine biofilm lysine and cadaverine contents before oral hygiene restriction (OHR), and their association with plaque index (PI) and gingival crevicular fluid (GCF) after OHR for a week. Methods Laser-induced fluorescence after capillary electrophoresis was used to determine lysine and cadaverine contents in dental biofilm, tongue biofilm and saliva before OHR and in dental biofilm after OHR. Results Before OHR, lysine and cadaverine contents of dental biofilm were similar and 10-fold greater than in saliva or tongue biofilm. After a week of OHR, the biofilm content of cadaverine increased and that of lysine decreased, consistent with greater biofilm lysine decarboxylase activity. Regression indicated that PI and GCF exudation were positively related to biofilm lysine post-OHR, unless biofilm lysine exceeded the minimal blood plasma content in which case PI was further increased but GCF exudation was reduced. Conclusions After OHR, lysine decarboxylase activity seems to determine biofilm lysine content and biofilm accumulation. When biofilm lysine exceeds minimal blood plasma content after OHR, less GCF appeared despite more biofilm. Lysine appears important for biofilm accumulation and the epithelial barrier to bacterial proinflammatory agents. Clinical Relevance Inhibiting lysine decarboxylase may retard the increased GCF exudation required for microbial development and gingivitis. PMID:22141361

  15. A molecular threading mechanism underlies Jumonji lysine demethylase KDM2A regulation of methylated H3K36

    PubMed Central

    Cheng, Zhongjun; Cheung, Peggie; Kuo, Alex J.; Yukl, Erik T.; Wilmot, Carrie M.

    2014-01-01

    The dynamic reversible methylation of lysine residues on histone proteins is central to chromatin biology. Key components are demethylase enzymes, which remove methyl moieties from lysine residues. KDM2A, a member of the Jumonji C domain-containing histone lysine demethylase family, specifically targets lower methylation states of H3K36. Here, structural studies reveal that H3K36 specificity for KDM2A is mediated by the U-shaped threading of the H3K36 peptide through a catalytic groove within KDM2A. The side chain of methylated K36 inserts into the catalytic pocket occupied by Ni2+ and cofactor, where it is positioned and oriented for demethylation. Key residues contributing to K36me specificity on histone H3 are G33 and G34 (positioned within a narrow channel), P38 (a turn residue), and Y41 (inserts into its own pocket). Given that KDM2A was found to also bind the H3K36me3 peptide, we postulate that steric constraints could prevent α-ketoglutarate from undergoing an “off-line”-to-“in-line” transition necessary for the demethylation reaction. Furthermore, structure-guided substitutions of residues in the KDM2A catalytic pocket abrogate KDM2A-mediated functions important for suppression of cancer cell phenotypes. Together, our results deduce insights into the molecular basis underlying KDM2A regulation of the biologically important methylated H3K36 mark. PMID:25128496

  16. Plasmodium falciparum Sir2A preferentially hydrolyzes medium and long chain fatty acyl lysine

    PubMed Central

    Zhu, Anita Y.; Zhou, Yeyun; Khan, Saba; Deitsch, Kirk W.; Hao, Quan; Lin, Hening

    2011-01-01

    Plasmodium falciparum Sir2A (PfSir2A), a member of the sirtuin family of nicotinamide adenine dinucleotide-dependent deacetylases, has been shown to regulate the expression of surface antigens to evade the detection by host immune surveillance. It is thought that PfSir2A achieves this by deacetylating histones. However, the deacetylase activity of PfSir2A is weak. Here we present enzymology and structural evidences supporting that PfSir2A catalyzes the hydrolysis of medium and long chain fatty acyl groups from lysine residues more efficiently. Furthermore, P. falciparum proteins are found to contain such fatty acyl lysine modifications that can be removed by purified PfSir2A in vitro. Together, the data suggest that the physiological function of PfSir2A in antigen variation may be achieved by removing medium and long chain fatty acyl groups from protein lysine residues. The robust activity of PfSir2A would also facilitate the development of PfSir2A inhibitors, which may have therapeutic value in malaria treatment. PMID:21992006

  17. Acetyl-lysine erasers and readers in the control of pulmonary hypertension and right ventricular hypertrophy

    PubMed Central

    Stratton, Matthew S.; McKinsey, Timothy A.

    2016-01-01

    Acetylation of lysine residues within nucleosomal histone tails provides a crucial mechanism for epigenetic control of gene expression. Acetyl groups are coupled to lysine residues by histone acetyltransferases (HATs) and removed by histone deacetylases (HDACs), which are also commonly referred to as “writers” and “erasers”, respectively. In addition to altering the electrostatic properties of histones, lysine acetylation often creates docking sites for bromodomain-containing “reader” proteins. This review focuses on epigenetic control of pulmonary hypertension (PH) and associated right ventricular (RV) cardiac hypertrophy and failure. Effects of small molecule HDAC inhibitors in pre-clinical models of PH are highlighted. Furthermore, we describe the recently discovered role of bromodomain and extraterminal (BET) reader proteins in the control of cardiac hypertrophy, and provide evidence suggesting that one member of this family, BRD4, contributes to the pathogenesis of RV failure. Together, the data suggest intriguing potential for pharmacological epigenetic therapies for the treatment of PH and right-sided heart failure. PMID:25707943

  18. Improved L-lysine production with Corynebacterium glutamicum and systemic insight into citrate synthase flux and activity.

    PubMed

    van Ooyen, Jan; Noack, Stephan; Bott, Michael; Reth, Alexander; Eggeling, Lothar

    2012-08-01

    We here developed a series of Corynebacterium glutamicum strains with gradual decreased specific citrate synthase (CS) activity and quantified in a multifaceted approach the consequences of residual activity on the transcriptome, metabolome, and fluxome level as well as on L-lysine formation and growth. We achieved an intended gradual L-lysine yield increase and recognized and overcame further new limitations in the L-lysine biosynthesis pathway to result in a strain with the highest yield reported so far when assayed under comparable conditions. As a non-intended outcome, a detailed flux analysis revealed an almost constant flux through CS at 10% remaining CS activity, whereas the metabolome data revealed an increase in the oxaloacetate and acetyl-CoA concentrations. Hence reduced CS activity is apparently efficiently buffered by increased concentrations of CS substrates, implying a certain robustness of the central metabolism in response of the imposed gene expressions. PMID:22392073

  19. Template-nucleated alanine-lysine helices are stabilized by position-dependent interactions between the lysine side chain and the helix barrel.

    PubMed

    Groebke, K; Renold, P; Tsang, K Y; Allen, T J; McClure, K F; Kemp, D S

    1996-04-30

    The helicity in water has been determined for several series of alanine-rich peptides that contain single lysine residues and that are N-terminally linked to a helix-inducing and reporting template termed Ac-Hel1. The helix-propagating constant for alanine (sAla value) that best fits the properties of these peptides lies in the range of 1.01-1.02, close to the value reported by Scheraga and coworkers [Wojcik, J., Altmann, K.-H. & Scheraga, H.A. (1990) Biopolymers 30, 121-134], but significantly lower than the value assigned by Baldwin and coworkers [Chakrabartty, A., Kortemme, T. & Baldwin, R.L. (1994) Protein Sci. 3,843-852]. From a study of conjugates Ac-Hel1-Ala(n)-Lys-Ala(m)-NH2 and analogs in which the methylene portion of the lysine side chain is truncated, we find that the unusual helical stability of Ala(n)Lys peptides is controlled primarily by interactions of the lysine side chain with the helix barrel, and only passively by the alanine matrix. Using 1H NMR spectroscopy, we observe nuclear Overhauser effect crosspeaks consistent with proton-proton contacts expected for these interactions. PMID:8633010

  20. N epsilon,N epsilon-dimethyl-lysine cytochrome c as an NMR probe for lysine involvement in protein-protein complex formation.

    PubMed Central

    Moore, G R; Cox, M C; Crowe, D; Osborne, M J; Rosell, F I; Bujons, J; Barker, P D; Mauk, M R; Mauk, A G

    1998-01-01

    The reductively dimethylated derivatives of horse and yeast iso-1-ferricytochromes c have been prepared and characterized for use as NMR probes of the complexes formed by cytochrome c with bovine liver cytochrome b5 and yeast cytochrome c peroxidase. The electrostatic properties and structures of the derivatized cytochromes are not significantly perturbed by the modifications; neither are the electrostatics of protein-protein complex formation or rates of interprotein electron transfer. Two-dimensional 1H-13C NMR spectroscopy of the complexes formed by the derivatized cytochromes with cytochrome b5 and cytochrome c peroxidase has been used to investigate the number and identity of lysine residues of cytochrome c that are involved in interprotein interactions of cytochrome c. The NMR data are incompatible with simple static models proposed previously for the complexes formed by these proteins, but are consistent with the presence of multiple, interconverting complexes of comparable stability, consistent with studies employing Brownian dynamics to model the complexes. The NMR characteristics of the Nepsilon,Nepsilon-dimethyl-lysine groups, their chemical shift dispersion, oxidation state and temperature dependences and the possibility of chemical exchange phenomena are discussed with relevance to the utility of Nepsilon, Nepsilon-dimethyl-lysine's being a generally useful derivative for characterizing protein-protein complexes. PMID:9601073

  1. KDM1 histone lysine demethylases as targets for treatments of oncological and neurodegenerative disease.

    PubMed

    Maes, Tamara; Mascaró, Cristina; Ortega, Alberto; Lunardi, Serena; Ciceri, Filippo; Somervaille, Tim C P; Buesa, Carlos

    2015-01-01

    Histone methylation and demethylation are important processes associated with the regulation of gene transcription, and alterations in histone methylation status have been linked to a large number of human diseases. Initially thought to be an irreversible process, histone methylation is now known to be reversed by two families of proteins containing over 30 members that act to remove methyl groups from specific lysine residues present in the tails of histone H3 and histone H4. A rapidly growing number of reports have implicated the FAD-dependent lysine specific demethylase (KDM1) family in cancer, and several small-molecule inhibitors are in development for the treatment of cancer. An additional role has emerged for KDM1 in brain function, offering additional opportunities for the development of novel therapeutic strategies in neurodegenerative disease. A decade after the identification of KDM1A as a histone demethylase, the first selective inhibitors have now reached the clinic. PMID:26111032

  2. Bromodomains: Translating the words of lysine acetylation into myelin injury and repair.

    PubMed

    Ntranos, Achilles; Casaccia, Patrizia

    2016-06-20

    Bromodomains are evolutionarily highly conserved α-helical structural motifs that recognize and bind acetylated lysine residues. Lysine acetylation is being increasingly recognized as a major posttranslational modification involved in diverse cellular processes and protein interactions and its deregulation has been implicated in the pathophysiology of various human diseases, such as multiple sclerosis and cancer. Bromodomain-containing proteins can have a wide variety of functions, ranging from histone acetyltransferase activity and chromatin remodeling to transcriptional mediation and co-activation. The role of bromodomains in translating a deregulated cell acetylome into disease phenotypes was recently unveiled by the development of small molecule bromodomain inhibitors. This breakthrough discovery highlighted bromodomain-containing proteins as key players of inflammatory pathways responsible for myelin injury and also demonstrated their role in several aspects of myelin repair including oligodendrocyte differentiation and axonal regeneration. PMID:26472704

  3. The L3MBTL3 Methyl-Lysine Reader Domain Functions As a Dimer.

    PubMed

    Baughman, Brandi M; Pattenden, Samantha G; Norris, Jacqueline L; James, Lindsey I; Frye, Stephen V

    2016-03-18

    L3MBTL3 recognizes mono- and dimethylated lysine residues on histone tails. The recently reported X-ray cocrystal structures of the chemical probe UNC1215 and inhibitor UNC2533 bound to the methyl-lysine reading MBT domains of L3MBTL3 demonstrate a unique and flexible 2:2 dimer mode of recognition. In this study, we describe our in vitro analysis of L3MBTL3 dimerization via its MBT domains and additionally show that this dimerization occurs within a cellular context in the absence of small molecule ligands. Furthermore, mutations to the first and second MBT domains abrogated L3MBTL3 dimerization both in vitro and in cells. These observations are consistent with the hypothesis that L3MBTL3 engages methylated histone tails as a dimer while carrying out its normal function and provides an explanation for the presence of repeated MBT domains within L3MBTL3. PMID:26317848

  4. Purification, Biochemical Analysis, and Structure Determination of JmjC Lysine Demethylases.

    PubMed

    Krishnan, S; Trievel, R C

    2016-01-01

    Jumonji C (JmjC) lysine demethylases (KDMs) catalyze the site- and state-specific demethylation of lysine residues in histone and nonhistone protein substrates. These enzymes have been implicated in diverse genomic processes, including epigenetic gene regulation, DNA damage response, DNA replication, and regulation of heterochromatin structure. In addition, a number of JmjC KDMs contribute to the incidence of numerous cancers, rendering them targets for the development of novel chemotherapeutic drugs. Using the JMJD2 KDM subfamily as representative examples, this chapter outlines strategies for purifying highly active, recombinant JmjC KDMs lacking inhibitory transition metal ions, characterizing kinetic parameters of these enzymes using a coupled fluorescent assay, and determining crystal structures of the enzymes in complex with methylated histone peptides. Together, these approaches provide a foundation for structural and biochemical characterization of the JmjC KDMs and facilitate efforts to identify small molecule inhibitors through high-throughput screening and structure-guided design. PMID:27372758

  5. Dynamic Motion and Communication in the Streptococcal C1 Phage Lysin, PlyC

    PubMed Central

    Reboul, Cyril F.; Cowieson, Nathan P.; Costa, Mauricio G. S.; Kass, Itamar; Jackson, Colin; Perahia, David; Buckle, Ashley M.; McGowan, Sheena

    2015-01-01

    The growing problem of antibiotic resistance underlies the critical need to develop new treatments to prevent and control resistant bacterial infection. Exogenous application of bacteriophage lysins results in rapid and specific destruction of Gram-positive bacteria and therefore lysins represent novel antibacterial agents. The PlyC phage lysin is the most potent lysin characterized to date and can rapidly lyse Group A, C and E streptococci. Previously, we have determined the X-ray crystal structure of PlyC, revealing a complicated and unique arrangement of nine proteins. The scaffold features a multimeric cell-wall docking assembly bound to two catalytic domains that communicate and work synergistically. However, the crystal structure appeared to be auto-inhibited and raised important questions as to the mechanism underlying its extreme potency. Here we use small angle X-ray scattering (SAXS) and reveal that the conformational ensemble of PlyC in solution is different to that in the crystal structure. We also investigated the flexibility of the enzyme using both normal mode (NM) analysis and molecular dynamics (MD) simulations. Consistent with our SAXS data, MD simulations show rotational dynamics of both catalytic domains, and implicate inter-domain communication in achieving a substrate-ready conformation required for enzyme function. Our studies therefore provide insights into how the domains in the PlyC holoenzyme may act together to achieve its extraordinary potency. PMID:26470022

  6. Critical body residues, Michaelis-Menten analysis of bioaccumulation, lethality and behaviour as endpoints of waterborne Ni toxicity in two teleosts.

    PubMed

    Leonard, Erin M; Marentette, Julie R; Balshine, Sigal; Wood, Chris M

    2014-03-01

    Traditionally, water quality guidelines/criteria are based on lethality tests where results are expressed as a function of waterborne concentrations (e.g. LC50). However, there is growing interest in the use of uptake and binding relationships, such as biotic ligand models (BLM), and in bioaccumulation parameters, such as critical body residue values (e.g. CBR50), to predict metal toxicity in aquatic organisms. Nevertheless, all these approaches only protect species against physiological death (e.g. mortality, failed recruitment), and do not consider ecological death which can occur at much lower concentrations when the animal cannot perform normal behaviours essential for survival. Therefore, we investigated acute (96 h) Ni toxicity in two freshwater fish species, the round goby (Neogobius melanostomus) and rainbow trout (Oncorhynchus mykiss) and compared LC, BLM, and CBR parameters for various organs, as well as behavioural responses (spontaneous activity). In general, round goby were more sensitive. Ni bioaccumulation displayed Michaelis-Menten kinetics in most tissues, and round goby gills had lower Kd (higher binding affinity) but similar Bmax (binding site density) values relative to rainbow trout gills. Round goby also accumulated more Ni than did trout in most tissues at a given exposure concentration. Organ-specific 96 h acute CBR values tended to be higher in round goby but 96 h acute CBR50 and CBR10 values in the gills were very similar in the two species. In contrast, LC50 and LC10 values were significantly higher in rainbow trout. With respect to BLM parameters, gill log KNiBL values for bioaccumulation were higher by 0.4-0.8 log units than the log KNiBL values for toxicity in both species, and both values were higher in goby (more sensitive). Round goby were also more sensitive with respect to the behavioural response, exhibiting a significant decline of 63-75 % in movements per minute at Ni concentrations at and above only 8 % of the LC50 value

  7. Determining high-quality critical body residues for multiple species and chemicals by applying improved experimental design and data interpretation concepts.

    PubMed

    van der Heijden, Stephan A; Hermens, Joop L M; Sinnige, Theo L; Mayer, Philipp; Gilbert, Dorothea; Jonker, Michiel T O

    2015-02-01

    Ecotoxicological effect data are generally expressed as effective concentrations in the external exposure medium and do thus not account for differences in chemical uptake, bioavailability, and metabolism, which can introduce substantial data variation. The Critical Body Residue (CBR) concept provides clear advantages, because it links effects directly to the internal exposure. Using CBRs instead of external concentrations should therefore reduce variability. For compounds that act via narcosis even a constant CBR has been proposed. Despite the expected uniformity, CBR values for these compounds still show large variability, possibly due to biased and inconsistent experimental testing. In the present study we tested whether variation in CBR data can be substantially reduced when using an improved experimental design and avoiding confounding factors. The aim was to develop and apply a well-defined test protocol for accurately and precisely measuring CBR data, involving improved (passive) dosing, sampling, and processing of organisms. The chemicals 1,2,4-trichlorobenzene, 1,2,3,4-tetrachlorobenzene, 2,3,4-trichloroaniline, 2,3,5,6-tetrachloroaniline, 4-chloro-3-methylphenol, pentylbenzene, pyrene, and bromophos-methyl were tested on Lumbriculus variegatus (California blackworm), Hyalella azteca (scud), and Poecilia reticulata (guppy), which yielded a high-quality database of 348 individual CBR values. Medians of CBR values ranged from 2.1 to 16.1 mmol/kg wet weight (ww) within all combinations of chemicals and species, except for the insecticide bromophos-methyl, for which the median was 1.3 mmol/kg ww. The new database thus covers about one log unit, which is considerably less than in existing databases. Medians differed maximally by a factor of 8.4 between the 7 chemicals but within one species, and by a factor of 2.6 between the three species but for individual chemicals. Accounting for the chemicals' internal distribution to different partitioning domains and

  8. Toxicity of lead and zinc to developing mussel and sea urchin embryos: critical tissue residues and effects of dissolved organic matter and salinity.

    PubMed

    Nadella, Sunita R; Tellis, Margaret; Diamond, Rachael; Smith, Scott; Bianchini, Adalto; Wood, Chris M

    2013-08-01

    Lead (Pb) EC50 values in the very sensitive early development phases (48-72h post-fertilization) of the mussels Mytilus galloprovincialis and Mytilus trossolus and sea urchin Strongylocentrotus purpuratus in 100% sea water were: M. trossolus - 45 (95% C.I.=22-72) μgL(-1); M. galloprovincialis - 63 (36-94) μgL(-1); S. purpuratus - 74 (50-101) μgL(-1). Salinity thresholds for normal development varied: M. trossolus>21ppt; M. galloprovincialis>28ppt; S. purpuratus≥30ppt. Addition of two spectroscopically distinct dissolved organic matters (DOM) from fresh water (Nordic Reservoir) and sea water (Inshore) moderately decreased the toxicity of Pb to both mussels, but not in a concentration-dependent fashion, with only an approximate doubling of EC50 over the range of 1.4-11.2mgCL(-1). Independent Pb binding capacity determinations for DOC explained the lack of a relationship between DOM concentration and toxicity. Salinity had no effect on Pb toxicity down to 21ppt in M. trossolus, and low salinity (21ppt) did not enhance the protective effect of DOC. Both DOMs increased the toxicity of Pb in developing sea urchin embryos, in contrast to mussels. Relative to Pb, the organisms were 6-9 fold less sensitive to Zn on a molar basis in 100% seawater with the following Zn EC50s: M. trossolus - 135 (103-170) μgL(-1); M. galloprovincialis - 172 (126-227) μgL(-1), S. purpuratus - 151 (129-177) μgL(-1). Nordic Reservoir and Inshore DOM (2-12mgCL(-1)) had no significant effect on Zn toxicity to mussels, in accord with voltammetry data showing an absence of any strong ligand binding for Zn by DOMs. As with Pb, DOMs increased Zn toxicity to urchin larvae. Critical Tissue Residues (CTR) based on whole body concentrations of Pb and Zn were determined for M. galloprovincialis at 48h and S. purpuratus at 72h. The median lethal CTR values (LA50s), useful parameters for development of saltwater Biotic Ligand Models (BLMs), were approximately 4-fold higher on a molar basis for Zn than

  9. Hydroxyphenylation of Histone Lysines: Post-translational Modification by Quinone Imines.

    PubMed

    Ravindra, Kodihalli C; Trudel, Laura J; Wishnok, John S; Wogan, Gerald N; Tannenbaum, Steven R; Skipper, Paul L

    2016-05-20

    Monocyclic aromatic amines are widespread environmental contaminants with multiple sources such as combustion products, pharmaceuticals, and pesticides. Their phenolic metabolites are converted intracellularly to electrophilic quinone imines upon autoxidation and can embed in the cellular matrix through a transimination reaction that leaves a redox-active residue as a substituent of lysine side-chain amino groups. To demonstrate the occurrence of this process within the cellular nucleus, Chinese hamster ovary AA8 cells were treated with the para-phenol of 3,5-dimethylamine, after which the histone proteins were isolated, derivatized, and subjected to tryptic digestion. The resulting peptides were analyzed by tandem mass spectrometry to determine which lysines were modified. Nine residues in histones H2A, H2B, and H4 were identified; these were located in histone tails, close to where DNA makes contact with the nuclear core particle, elsewhere on the protein surface, and deep within the core. Kinetics of disappearance of the modified lysines in cultured cells was determined using isotope-dilution mass spectrometry. AA8 cells were also transfected with the genetically encoded hydrogen peroxide biosensor HyPer in constructs that lead to expression of HyPer in different cellular compartments. Challenging the resulting cells with the dimethylaminophenol resulted in sustained fluorescence emission in each of the compartments, demonstrating ongoing production of H2O2. The kinetics of modified lysine loss determined by mass spectrometry was consistent with persistence of HyPer fluorescence emission. We conclude that the para-phenol of 3,5-dimethylamine can become stably integrated into the histone proteins, which are minimally repaired, if at all, and function as a persistent source of intracellular H2O2. PMID:26866676

  10. Exploring lysine riboswitch for metabolic flux control and improvement of L-lysine synthesis in Corynebacterium glutamicum.

    PubMed

    Zhou, Li-Bang; Zeng, An-Ping

    2015-06-19

    Riboswitch, a regulatory part of an mRNA molecule that can specifically bind a metabolite and regulate gene expression, is attractive for engineering biological systems, especially for the control of metabolic fluxes in industrial microorganisms. Here, we demonstrate the use of lysine riboswitch and intracellular l-lysine as a signal to control the competing but essential metabolic by-pathways of lysine biosynthesis. To this end, we first examined the natural lysine riboswitches of Eschericia coli (ECRS) and Bacillus subtilis (BSRS) to control the expression of citrate synthase (gltA) and thus the metabolic flux in the tricarboxylic acid (TCA) cycle in E. coli. ECRS and BSRS were then successfully used to control the gltA gene and TCA cycle activity in a lysine producing strain Corynebacterium glutamicum LP917, respectively. Compared with the strain LP917, the growth of both lysine riboswitch-gltA mutants was slower, suggesting a reduced TCA cycle activity. The lysine production was 63% higher in the mutant ECRS-gltA and 38% higher in the mutant BSRS-gltA, indicating a higher metabolic flux into the lysine synthesis pathway. This is the first report on using an amino acid riboswitch for improvement of lysine biosynthesis. The lysine riboswitches can be easily adapted to dynamically control other essential but competing metabolic pathways or even be engineered as an "on-switch" to enhance the metabolic fluxes of desired metabolic pathways. PMID:25575181

  11. Examining the critical roles of human CB2 receptor residues Valine 3.32 (113) and Leucine 5.41 (192) in ligand recognition and downstream signaling activities.

    PubMed

    Alqarni, Mohammed; Myint, Kyaw Zeyar; Tong, Qin; Yang, Peng; Bartlow, Patrick; Wang, Lirong; Feng, Rentian; Xie, Xiang-Qun

    2014-09-26

    We performed molecular modeling and docking to predict a putative binding pocket and associated ligand-receptor interactions for human cannabinoid receptor 2 (CB2). Our data showed that two hydrophobic residues came in close contact with three structurally distinct CB2 ligands: CP-55,940, SR144528 and XIE95-26. Site-directed mutagenesis experiments and subsequent functional assays implicated the roles of Valine residue at position 3.32 (V113) and Leucine residue at position 5.41 (L192) in the ligand binding function and downstream signaling activities of the CB2 receptor. Four different point mutations were introduced to the wild type CB2 receptor: V113E, V113L, L192S and L192A. Our results showed that mutation of Val113 with a Glutamic acid and Leu192 with a Serine led to the complete loss of CB2 ligand binding as well as downstream signaling activities. Substitution of these residues with those that have similar hydrophobic side chains such as Leucine (V113L) and Alanine (L192A), however, allowed CB2 to retain both its ligand binding and signaling functions. Our modeling results validated by competition binding and site-directed mutagenesis experiments suggest that residues V113 and L192 play important roles in ligand binding and downstream signaling transduction of the CB2 receptor. PMID:25148941

  12. Protein lysine acetylation in bacteria: Current state of the art.

    PubMed

    Ouidir, Tassadit; Kentache, Takfarinas; Hardouin, Julie

    2016-01-01

    Post-translational modifications of proteins are key events in cellular metabolism and physiology regulation. Lysine acetylation is one of the best studied protein modifications in eukaryotes, but, until recently, ignored in bacteria. However, proteomic advances have highlighted the diversity of bacterial lysine-acetylated proteins. The current data support the implication of lysine acetylation in various metabolic pathways, adaptation and virulence. In this review, we present a broad overview of the current knowledge of lysine acetylation in bacteria. We emphasize particularly the significant contribution of proteomics in this field. PMID:26390373

  13. Global analysis of lysine acetylation in strawberry leaves

    PubMed Central

    Fang, Xianping; Chen, Wenyue; Zhao, Yun; Ruan, Songlin; Zhang, Hengmu; Yan, Chengqi; Jin, Liang; Cao, Lingling; Zhu, Jun; Ma, Huasheng; Cheng, Zhongyi

    2015-01-01

    Protein lysine acetylation is a reversible and dynamic post-translational modification. It plays an important role in regulating diverse cellular processes including chromatin dynamic, metabolic pathways, and transcription in both prokaryotes and eukaryotes. Although studies of lysine acetylome in plants have been reported, the throughput was not high enough, hindering the deep understanding of lysine acetylation in plant physiology and pathology. In this study, taking advantages of anti-acetyllysine-based enrichment and high-sensitive-mass spectrometer, we applied an integrated proteomic approach to comprehensively investigate lysine acetylome in strawberry. In total, we identified 1392 acetylation sites in 684 proteins, representing the largest dataset of acetylome in plants to date. To reveal the functional impacts of lysine acetylation in strawberry, intensive bioinformatic analysis was performed. The results significantly expanded our current understanding of plant acetylome and demonstrated that lysine acetylation is involved in multiple cellular metabolism and cellular processes. More interestingly, nearly 50% of all acetylated proteins identified in this work were localized in chloroplast and the vital role of lysine acetylation in photosynthesis was also revealed. Taken together, this study not only established the most extensive lysine acetylome in plants to date, but also systematically suggests the significant and unique roles of lysine acetylation in plants. PMID:26442052

  14. ε-Poly-L-lysine peptide chain length regulated by the linkers connecting the transmembrane domains of ε-Poly-L-lysine synthetase.

    PubMed

    Hamano, Yoshimitsu; Kito, Naoko; Kita, Akihiro; Imokawa, Yuuki; Yamanaka, Kazuya; Maruyama, Chitose; Katano, Hajime

    2014-08-01

    ε-Poly-l-lysine (ε-PL), consisting of 25 to 35 l-lysine residues with linkages between the α-carboxyl groups and ε-amino groups, is produced by Streptomyces albulus NBRC14147. ε-PL synthetase (Pls) is a membrane protein with six transmembrane domains (TM1 to TM6) as well as both an adenylation domain and a thiolation domain, characteristic of the nonribosomal peptide synthetases. Pls directly generates ε-PL chain length diversity (25- to 35-mer), but the processes that control the chain length of ε-PL during the polymerization reaction are still not fully understood. Here, we report on the identification of Pls amino acid residues involved in the regulation of the ε-PL chain length. From approximately 12,000 variants generated by random mutagenesis, we found 8 Pls variants that produced shorter chains of ε-PL. These variants have one or more mutations in two linker regions connecting the TM1 and TM2 domains and the TM3 and TM4 domains. In the Pls catalytic mechanism, the growing chain of ε-PL is not tethered to the enzyme, implying that the enzyme must hold the growing chain until the polymerization reaction is complete. Our findings reveal that the linker regions are important contributors to grasp the growing chain of ε-PL. PMID:24907331

  15. Enzymatic production of 5-aminovalerate from l-lysine using l-lysine monooxygenase and 5-aminovaleramide amidohydrolase

    PubMed Central

    Liu, Pan; Zhang, Haiwei; Lv, Min; Hu, Mandong; Li, Zhong; Gao, Chao; Xu, Ping; Ma, Cuiqing

    2014-01-01

    5-Aminovalerate is a potential C5 platform chemical for synthesis of valerolactam, 5-hydroxyvalerate, glutarate, and 1,5-pentanediol. It is a metabolite of l-lysine catabolism through the aminovalerate pathway in Pseudomonas putida. l-Lysine monooxygenase (DavB) and 5-aminovaleramide amidohydrolase (DavA) play key roles in the biotransformation of l-lysine into 5-aminovalerate. Here, DavB and DavA of P. putida KT2440 were expressed, purified, and coupled for the production of 5-aminovalerate from l-lysine. Under optimal conditions, 20.8 g/L 5-aminovalerate was produced from 30 g/L l-lysine in 12 h. Because l-lysine is an industrial fermentation product, the two-enzyme coupled system presents a promising alternative for the production of 5-aminovalerate. PMID:25012259

  16. Four basic residues critical for the ion selectivity and pore blocker sensitivity of TMEM16A calcium-activated chloride channels.

    PubMed

    Peters, Christian J; Yu, Haibo; Tien, Jason; Jan, Yuh Nung; Li, Min; Jan, Lily Yeh

    2015-03-17

    TMEM16A (transmembrane protein 16) (Anoctamin-1) forms a calcium-activated chloride channel (CaCC) that regulates a broad array of physiological properties in response to changes in intracellular calcium concentration. Although known to conduct anions according to the Eisenman type I selectivity sequence, the structural determinants of TMEM16A anion selectivity are not well-understood. Reasoning that the positive charges on basic residues are likely contributors to anion selectivity, we performed whole-cell recordings of mutants with alanine substitution for basic residues within the putative pore region and identified four residues on four different putative transmembrane segments that significantly increased the permeability of the larger halides and thiocyanate relative to that of chloride. Because TMEM16A permeation properties are known to shift with changes in intracellular calcium concentration, we further examined the calcium dependence of anion selectivity. We found that WT TMEM16A but not mutants with alanine substitution at those four basic residues exhibited a clear decline in the preference for larger anions as intracellular calcium was increased. Having implicated these residues as contributing to the TMEM16A pore, we scrutinized candidate small molecules from a high-throughput CaCC inhibitor screen to identify two compounds that act as pore blockers. Mutations of those four putative pore-lining basic residues significantly altered the IC50 of these compounds at positive voltages. These findings contribute to our understanding regarding anion permeation of TMEM16A CaCC and provide valuable pharmacological tools to probe the channel pore. PMID:25733897

  17. PR Domain-containing Protein 7 (PRDM7) Is a Histone 3 Lysine 4 Trimethyltransferase*

    PubMed Central

    Blazer, Levi L.; Lima-Fernandes, Evelyne; Gibson, Elisa; Eram, Mohammad S.; Loppnau, Peter; Arrowsmith, Cheryl H.; Schapira, Matthieu; Vedadi, Masoud

    2016-01-01

    PR domain-containing protein 7 (PRDM7) is a primate-specific histone methyltransferase that is the result of a recent gene duplication of PRDM9. The two proteins are highly homologous, especially in the catalytic PR/SET domain, where they differ by only three amino acid residues. Here we report that PRDM7 is an efficient methyltransferase that selectively catalyzes the trimethylation of H3 lysine 4 (H3K4) both in vitro and in cells. Through selective mutagenesis we have dissected the functional roles of each of the three divergent residues between the PR domains of PRDM7 and PRDM9. These studies indicate that after a single serine to tyrosine mutation at residue 357 (S357Y), PRDM7 regains the substrate specificities and catalytic activities similar to its evolutionary predecessor, including the ability to efficiently methylate H3K36. PMID:27129774

  18. Bacteriophage phi11 lysin: physicochemical characterization and comparison with phage phi80a lysin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phage lytic enzymes are promising antimicrobial agents. Lysins of phage phi11 (LysPhi11) and phi80a (LysPhi80a) can lyse (destroy) biofilms and cells of antibiotic-resistant strains of Staphylococcus aureus. Stability of enzymes is one of the parameters making their practical use possible. The obj...

  19. Automethylation of SUV39H2, an oncogenic histone lysine methyltransferase, regulates its binding affinity to substrate proteins.

    PubMed

    Piao, Lianhua; Nakakido, Makoto; Suzuki, Takehiro; Dohmae, Naoshi; Nakamura, Yusuke; Hamamoto, Ryuji

    2016-04-19

    We previously reported that the histone lysine methyltransferase SUV39H2, which is overexpressed in various types of human cancer, plays a critical role in the DNA repair after double strand breakage, and possesses oncogenic activity. Although its biological significance in tumorigenesis has been elucidated, the regulatory mechanism of SUV39H2 activity through post-translational modification is not well known. In this study, we demonstrate in vitro and in vivo automethylation of SUV39H2 at lysine 392. Automethylation of SUV39H2 led to impairment of its binding affinity to substrate proteins such as histone H3 and LSD1. Furthermore, we observed that hyper-automethylated SUV39H2 reduced methylation activities to substrates through affecting the binding affinity to substrate proteins. Our finding unveils a novel autoregulatory mechanism of SUV39H2 through lysine automethylation. PMID:26988914

  20. Automethylation of SUV39H2, an oncogenic histone lysine methyltransferase, regulates its binding affinity to substrate proteins

    PubMed Central

    Piao, Lianhua; Nakakido, Makoto; Suzuki, Takehiro; Dohmae, Naoshi; Nakamura, Yusuke; Hamamoto, Ryuji

    2016-01-01

    We previously reported that the histone lysine methyltransferase SUV39H2, which is overexpressed in various types of human cancer, plays a critical role in the DNA repair after double strand breakage, and possesses oncogenic activity. Although its biological significance in tumorigenesis has been elucidated, the regulatory mechanism of SUV39H2 activity through post-translational modification is not well known. In this study, we demonstrate in vitro and in vivo automethylation of SUV39H2 at lysine 392. Automethylation of SUV39H2 led to impairment of its binding affinity to substrate proteins such as histone H3 and LSD1. Furthermore, we observed that hyper-automethylated SUV39H2 reduced methylation activities to substrates through affecting the binding affinity to substrate proteins. Our finding unveils a novel autoregulatory mechanism of SUV39H2 through lysine automethylation. PMID:26988914

  1. the active site residue V266 of Chlamydial HtrA is critical for substrate binding during both in vitro and in vivo conditions.

    PubMed

    Gloeckl, Sarina; Tyndall, Joel D A; Stansfield, Scott H; Timms, Peter; Huston, Wilhelmina M

    2012-01-01

    HtrA is a complex, multimeric chaperone and serine protease important for the virulence and survival of many bacteria. Chlamydia trachomatis is an obligate, intracellular bacterial pathogen that is responsible for severe disease pathology. C. trachomatis HtrA (CtHtrA) has been shown to be highly expressed in laboratory models of disease. In this study, molecular modelling of CtHtrA protein active site structure identified putative S1-S3 subsite residues I242, I265, and V266. These residues were altered by site-directed mutagenesis, and these changes were shown to considerably reduce protease activity on known substrates and resulted in a narrower and distinct range of substrates compared to wild type. Bacterial two-hybrid analysis revealed that CtHtrA is able to interact in vivo with a broad range of protein sequences with high affinity. Notably, however, the interaction was significantly altered in 35 out of 69 clones when residue V266 was mutated, indicating that this residue has an important function during substrate binding. PMID:22353774

  2. 3-(Piperidin-4-ylmethoxy)pyridine Containing Compounds Are Potent Inhibitors of Lysine Specific Demethylase 1.

    PubMed

    Wu, Fangrui; Zhou, Chao; Yao, Yuan; Wei, Liping; Feng, Zizhen; Deng, Lisheng; Song, Yongcheng

    2016-01-14

    Methylation of histone lysine residues plays important roles in gene expression regulation as well as cancer initiation. Lysine specific demethylase 1 (LSD1) is responsible for maintaining balanced methylation levels at histone H3 lysine 4 (H3K4). LSD1 is a drug target for certain cancers, due to important functions of methylated H3K4 or LSD1 overexpression. We report the design, synthesis, and structure-activity relationships of 3-(piperidin-4-ylmethoxy)pyridine containing compounds as potent LSD1 inhibitors with Ki values as low as 29 nM. These compounds exhibited high selectivity (>160×) against related monoamine oxidase A and B. Enzyme kinetics and docking studies suggested they are competitive inhibitors against a dimethylated H3K4 substrate and provided a possible binding mode. The potent LSD1 inhibitors can increase cellular H3K4 methylation and strongly inhibit proliferation of several leukemia and solid tumor cells with EC50 values as low as 280 nM, while they had negligible effects on normal cells. PMID:26652247

  3. Lysines in the tetramerization domain of p53 selectively modulate G1 arrest.

    PubMed

    Beckerman, Rachel; Yoh, Kathryn; Mattia-Sansobrino, Melissa; Zupnick, Andrew; Laptenko, Oleg; Karni-Schmidt, Orit; Ahn, Jinwoo; Byeon, In-Ja; Keezer, Susan; Prives, Carol

    2016-06-01

    Functional in a tetrameric state, the protein product of the p53 tumor suppressor gene confers its tumor-suppressive activity by transactivating genes which promote cell-cycle arrest, senescence, or programmed cell death. How p53 distinguishes between these divergent outcomes is still a matter of considerable interest. Here we discuss the impact of 2 mutations in the tetramerization domain that confer unique properties onto p53. By changing lysines 351 and 357 to arginine, thereby blocking all post-translational modifications of these residues, DNA binding and transcriptional regulation by p53 remain virtually unchanged. On the other hand, by changing these lysines to glutamine (2KQ-p53), thereby neutralizing their positive charge and potentially mimicking acetylation, p53 is impaired in the induction of cell cycle arrest and yet can still effectively induce cell death. Surprisingly, when 2KQ-p53 is expressed at high levels in H1299 cells, it can bind to and transactivate numerous p53 target genes including p21, but not others such as miR-34a and cyclin G1 to the same extent as wild-type p53. Our findings show that strong induction of p21 is not sufficient to block H1299 cells in G1, and imply that modification of one or both of the lysines within the tetramerization domain may serve as a mechanism to shunt p53 from inducing cell cycle arrest. PMID:27210019

  4. Guanidination of Soluble Lysine-Rich Cyanophycin Yields a Homoarginine-Containing Polyamide

    PubMed Central

    Frommeyer, Maja; Bergander, Klaus

    2014-01-01

    Soluble cyanobacterial granule polypeptide (CGP), especially that isolated from recombinant Escherichia coli strains, consists of aspartic acid, arginine, and a greater amount of lysine than that in insoluble CGP isolated from cyanobacteria or various other recombinant bacteria. In vitro guanidination of lysine side chains of soluble CGP with o-methylisourea (OMIU) yielded the nonproteinogenic amino acid homoarginine. The modified soluble CGP consisted of 51 mol% aspartate, 14 mol% arginine, and 35 mol% homoarginine. The complete conversion of lysine residues to homoarginine was confirmed by (i) nuclear magnetic resonance spectrometry, (ii) coupled liquid chromatography-mass spectrometry, and (iii) high-performance liquid chromatography. Unlike soluble CGP, this new homoarginine-containing polyamide was soluble only under acidic or alkaline conditions and was insoluble in water or at a neutral pH. Thus, it showed solubility behavior similar to that of the natural insoluble polymer isolated from cyanobacteria, consisting of aspartic acid and arginine only. Polyacrylamide gel electrophoresis revealed similar degrees of polymerization of the native (12- to 40-kDa) and modified (10- to 35-kDa) polymers. This study showed that the chemical structure and properties of a biopolymer could be changed by in vitro introduction of a new functional group after biosynthesis of the native polymer. In addition, the modified CGP could be digested in vitro using the cyanophycinase from Pseudomonas alcaligenes strain DIP1, yielding a new dipeptide consisting of aspartate and homoarginine. PMID:24509932

  5. Reconstitution of active and stoichiometric multisubunit lysine acetyltransferase complexes in insect cells.

    PubMed

    Yan, Kezhi; Wu, Chao-Jung; Pelletier, Nadine; Yang, Xiang-Jiao

    2012-01-01

    Protein lysine acetyltransferases (KATs) catalyze acetylation of the ε-amino group on a specific lysine residue, and this posttranslational modification is important for regulating the function and activities of thousands of proteins in diverse organisms from bacteria to humans. Interestingly, many known KATs exist in multisubunit complexes and complex formation is important for their proper structure, function, and regulation. Thus, it is necessary to reconstitute enzymatically active complexes for studying the relationship between subunits and determining structures of the complexes. Due to inherent limitations of bacterial and mammalian expression systems, baculovirus-mediated protein expression in insect cells has proven useful for assembling such multisubunit complexes. Related to this, we have adopted such an approach for reconstituting active tetrameric complexes of monocytic leukemia zinc (MOZ, finger protein, recently renamed MYST3 or KAT6A) and MOZ-related factor (MORF, also known as MYST4 or KAT6B), two KATs directly linked to development of leukemia and self-renewal of stem cells. Herein, we use these complexes as examples to describe the related procedures. Similar methods have been used for reconstituting active complexes of histone deacetylases, lysine demethylases, and ubiquitin ligases, so this simple approach can be adapted for molecular dissection of various multisubunit complexes. PMID:22113293

  6. A stable phage lysin (Cpl-1) dimer with increased antipneumococcal activity and decreased plasma clearance.

    PubMed

    Resch, Gregory; Moreillon, Philippe; Fischetti, Vincent A

    2011-12-01

    Bacteriophages (phages) produce endolysins (lysins) as part of their lytic cycle in order to degrade the peptidoglycan layer of the infected bacteria for subsequent release of phage progeny. Because these enzymes maintain their lytic and lethal activity against Gram-positive bacteria when added extrinsically to the cells, they have been actively exploited as novel anti-infectives, sometimes termed enzybiotics. As with other relatively small peptides, one issue in their clinical development is their rapid inactivation through proteolytic degradation, immunological blockage and renal clearance. The antipneumococcal lysin Cpl-1 was shown to escape both proteolysis and immunological blockage. However, its short plasma half-life (20.5 min in mice) may represent a shortcoming for clinical usefulness. Here we report the construction of a Cpl-1 dimer with a view to increasing both the antipneumococcal specific activity and plasma half-life of Cpl-1. Dimerisation was achieved by introducing specific cysteine residues at the C-terminal end of the enzyme, thus favouring disulphide bonding. Compared with the native monomer, the constructed dimer demonstrated a two-fold increase in specific antipneumococcal activity and a ca. ten-fold decrease in plasma clearance. As several lysins are suspected to dimerise on contact with their cell wall substrate to be fully active, stable pre-dimerised enzymes may represent a more efficient alternative to the native monomer. PMID:21982146

  7. SUMOylation negatively modulates target gene occupancy of the KDM5B, a histone lysine demethylase.

    PubMed

    Bueno, Murilo T D; Richard, Stéphane

    2013-11-01

    The histone lysine demethylase KDM5B plays key roles in gene repression by demethylating trimethylated lysine 4 of histone H3 (H3K4me3), a modification commonly found at the promoter region of actively transcribed genes. KDM5B is known to regulate the expression of genes involved in cell cycle progression; however, little is known about the post-translational modifications that regulate KDM5B. Herein, we report that KDM5B is SUMOylated at lysine residues 242 and 278 and that the ectopic expression of the hPC2 SUMO E3 ligase enhances this SUMOylation. Interestingly, the levels of KDM5B and its SUMOylated forms are regulated during the cell cycle. KDM5B is modulated by RNF4, an E3 ubiquitin ligase that targets SUMO-modified proteins to proteasomal degradation. Digital gene expression analyses showed that cells expressing the SUMOylation-deficient KDM5B harbor repressed mRNA expression profiles of cell cycle and DNA repair genes. Chromatin immunoprecipitations confirmed some of these genes as KDM5B targets, as they displayed reduced H3K4me3 levels in cells ectopically expressing KDM5B. We propose that SUMOylation by hPC2 regulates the activity of KDM5B. PMID:23970103

  8. Significance of a negative exercise thallium test in the presence of a critical residual stenosis after thrombolysis for acute myocardial infarction

    SciTech Connect

    Sutton, J.M.; Topol, E.J. )

    1991-04-01

    After thrombolytic therapy for acute myocardial infarction, increasing emphasis is placed on early submaximal exercise testing, with further intervention advocated only for demonstrable ischemia. Although significant residual coronary artery lesions after successful thrombolysis are common, many patients paradoxically have no corresponding provokable ischemia. The relation between significant postthrombolytic residual coronary artery disease and a negative early, submaximal exercise thallium-201 tomogram was studied among 101 consecutive patients with uncomplicated myocardial infarction and at least 70% residual stenosis of the infarct artery. A negative test occurred in 49 (48.5%) patients with a mean 88% residual infarct artery stenosis. Further characteristics of the group were as follows: mean time to treatment was 3.1 hours; mean age was 54 +/- 10 years; 80% were male; 47% had anterior infarction; 39% had multivessel disease; mean left ventricular ejection fraction was 53 +/- 14%; and mean peak creatine kinase level was 3,820 +/- 3,123 IU/ml. A similar group of 52 (51.5%) patients, treated within 3.3 hours from symptom onset, with a mean postthrombolysis stenosis of 90%, had a positive exercise test. Characteristics of this group were as follows: age was 58 +/- 10 years; 92% were male; 56% had anterior infarction; 40% had multivessel disease; and mean left ventricular ejection fraction was 54 +/- 15%. The peak creatine kinase level associated with the infarction, however, was lower: 2,605 +/- 1,805 IU/ml (p = 0.04). There was no difference in performance at exercise testing with respect to peak systolic pressure, peak heart rate, or time tolerated on the treadmill between the two groups. By multivariate logistic regression, only peak creatine kinase level predicted a negative stress result in the presence of a significant residual stenosis.

  9. Inhibition of bactericidal and bacteriolytic activities of poly-D-lysine and lysozyme by chitotriose and ferric iron.

    PubMed Central

    Tompkins, G R; O'Neill, M M; Cafarella, T G; Germaine, G R

    1991-01-01

    In a previous report from this laboratory (N. J. Laible and G. R. Germaine, Infect. Immun. 48:720-728, 1985), evidence was presented to suggest that the bactericidal actions of both reduced (i.e., muramidase-inactive) human placental lysozyme and the synthetic cationic homopolymer poly-D-lysine involved the activation of a bacterial endogenous activity that was inhibitable by N,N',N"-triacetylchitotriose (chitotriose). In the present investigation however, we found that the bactericidal and bacteriolytic action of poly-D-lysine could be prevented only by some commercially available chitotriose preparations and not by others. Analysis by physical and chemical methods failed to distinguish protective chitotriose (CTa) and nonprotective chitotriose (CTi) preparations. CTi and CTa preparations displayed equal capacities to competitively inhibit binding of [3H]chitotriose by immobilized lysozyme and were indistinguishable in their abilities to block the lytic activity of lysozyme against Micrococcus lysodeikticus cells. Elemental analysis revealed significantly higher levels of phosphorus, calcium, iron, sodium, manganese, and copper in CTa. Removal of metals from CTa by chelate chromatography completely abolished the poly-D-lysine-protective capacity. Of the metals detected, only ferric iron (5 to 10 microM) mimicked the protective action of CTa. A Fe(III) concentration of 50 microM was required to inhibit lysozyme (5 micrograms/ml). Both Fe(III) and CTa (but not CTi) quantitatively blocked the labeling of poly-D-lysine by fluorescamine, suggesting that the primary amino groups of the lysine residues participate in iron binding. Thus, it appears that the poly-D-lysine-protective capacity of certain chitotriose preparations was due not to the chitotriose itself but to contaminating metal ions which interact directly with the polycationic agent. In contrast, Fe(III) cannot account for inhibition of either the bactericidal or bacteriolytic activity of lysozyme by

  10. Triple therapy with pyridoxine, arginine supplementation and dietary lysine restriction in pyridoxine-dependent epilepsy: Neurodevelopmental outcome.

    PubMed

    Coughlin, Curtis R; van Karnebeek, Clara D M; Al-Hertani, Walla; Shuen, Andrew Y; Jaggumantri, Sravan; Jack, Rhona M; Gaughan, Sommer; Burns, Casey; Mirsky, David M; Gallagher, Renata C; Van Hove, Johan L K

    2015-01-01

    Pyridoxine-dependent epilepsy (PDE) is an epileptic encephalopathy characterized by response to pharmacologic doses of pyridoxine. PDE is caused by deficiency of α-aminoadipic semialdehyde dehydrogenase resulting in impaired lysine degradation and subsequent accumulation of α-aminoadipic semialdehyde. Despite adequate seizure control with pyridoxine monotherapy, 75% of individuals with PDE have significant developmental delay and intellectual disability. We describe a new combined therapeutic approach to reduce putative toxic metabolites from impaired lysine metabolism. This approach utilizes pyridoxine, a lysine-restricted diet to limit the substrate that leads to neurotoxic metabolite accumulation and L-arginine to compete for brain lysine influx and liver mitochondrial import. We report the developmental and biochemical outcome of six subjects who were treated with this triple therapy. Triple therapy reduced CSF, plasma, and urine biomarkers associated with neurotoxicity in PDE. The addition of arginine supplementation to children already treated with dietary lysine restriction and pyridoxine further reduced toxic metabolites, and in some subjects appeared to improve neurodevelopmental outcome. Dietary lysine restriction was associated with improved seizure control in one subject, and the addition of arginine supplementation increased the objective motor outcome scale in two twin siblings, illustrating the contribution of each component of this treatment combination. Optimal results were noted in the individual treated with triple therapy early in the course of the disease. Residual disease symptoms could be related to early injury suggested by initial MR imaging prior to initiation of treatment or from severe epilepsy prior to diagnosis. This observational study reports the use of triple therapy, which combines three effective components in this rare condition, and suggests that early diagnosis and treatment with this new triple therapy may ameliorate the

  11. Inhibition of bactericidal and bacteriolytic activities of poly-D-lysine and lysozyme by chitotriose and ferric iron.

    PubMed

    Tompkins, G R; O'Neill, M M; Cafarella, T G; Germaine, G R

    1991-02-01

    In a previous report from this laboratory (N. J. Laible and G. R. Germaine, Infect. Immun. 48:720-728, 1985), evidence was presented to suggest that the bactericidal actions of both reduced (i.e., muramidase-inactive) human placental lysozyme and the synthetic cationic homopolymer poly-D-lysine involved the activation of a bacterial endogenous activity that was inhibitable by N,N',N"-triacetylchitotriose (chitotriose). In the present investigation however, we found that the bactericidal and bacteriolytic action of poly-D-lysine could be prevented only by some commercially available chitotriose preparations and not by others. Analysis by physical and chemical methods failed to distinguish protective chitotriose (CTa) and nonprotective chitotriose (CTi) preparations. CTi and CTa preparations displayed equal capacities to competitively inhibit binding of [3H]chitotriose by immobilized lysozyme and were indistinguishable in their abilities to block the lytic activity of lysozyme against Micrococcus lysodeikticus cells. Elemental analysis revealed significantly higher levels of phosphorus, calcium, iron, sodium, manganese, and copper in CTa. Removal of metals from CTa by chelate chromatography completely abolished the poly-D-lysine-protective capacity. Of the metals detected, only ferric iron (5 to 10 microM) mimicked the protective action of CTa. A Fe(III) concentration of 50 microM was required to inhibit lysozyme (5 micrograms/ml). Both Fe(III) and CTa (but not CTi) quantitatively blocked the labeling of poly-D-lysine by fluorescamine, suggesting that the primary amino groups of the lysine residues participate in iron binding. Thus, it appears that the poly-D-lysine-protective capacity of certain chitotriose preparations was due not to the chitotriose itself but to contaminating metal ions which interact directly with the polycationic agent. In contrast, Fe(III) cannot account for inhibition of either the bactericidal or bacteriolytic activity of lysozyme by

  12. System-wide Studies of N-Lysine Acetylation in Rhodopseudomonas palustris Reveals Substrate Specificity of Protein Acetyltransferases

    SciTech Connect

    Crosby, Heidi A; Pelletier, Dale A; Hurst, Gregory {Greg} B; Escalante-Semerena, Jorge C

    2012-01-01

    Background: Protein acetylation is widespread in prokaryotes. Results: Six new acyl-CoA synthetases whose activities are controlled by acetylation were identified, and their substrate preference established. A new protein acetyltransferase was also identified and its substrate specificity determined. Conclusion: Protein acetyltransferases acetylate a conserved lysine residue in protein substrates. Significance: The R. palustris Pat enzyme specifically acetylates AMP-forming acyl-CoA synthetases and regulates fatty acid metabolism.

  13. Comparison of magnetic carboxymethyl chitosan nanoparticles and cation exchange resin for the efficient purification of lysine-tagged small ubiquitin-like modifier protease.

    PubMed

    Li, Junhua; Zhang, Yang; Shen, Fei; Yang, Yanjun

    2012-10-15

    A fusion tag that can be purified by the cheap ion-exchanger based on the ionic binding force may provide a cost-effective scheme over other affinity fusion tags. Small ubiquitin-like modifier (SUMO) protease derived from Saccharomyces cerevisiae was fused with a poly lysine tag containing 10 lysine residues at its C-terminus and then expressed in Escherichia coli. The ionic binding force provided by the ploy lysine tag allowed the selective recovery of the small ubiquitin-like modifier protease from recombinant E. coli cell extracts. A preliminary comparative study of the adsorption and elution of poly lysine tagged SUMO protease on Amberlite Cobalamion and magnetite carboxymethyl chitosan nanoparticles was performed. Amberlite Cobalamion and magnetite nanoparticles had the similar elution profile due to the common functional groups - carboxyl groups. The maximum dynamic adsorption capacity of Amberlite Cobalamion and magnetite nanoparticles reached 36.8 and 211.4 mg/g, respectively. The lysine-tagged protease can be simply purified by magnetite nanoparticles from cell extracts with higher purity than that by Amberlite Cobalamion. The superparamagnetic nanoparticles possess the advantages of highly specific, fast and excellent binding of a larger amount of lysine tagged SUMO modifier protease, and it is also easier to separate from the crude biological process liquors compared with the conventional separation techniques of polycationic amino acids fusion proteins. PMID:22995375

  14. Enhancement of lysine acetylation accelerates wound repair

    PubMed Central

    Spallotta, Francesco; Cencioni, Chiara; Straino, Stefania; Sbardella, Gianluca; Castellano, Sabrina; Capogrossi, Maurizio C; Martelli, Fabio; Gaetano, Carlo

    2013-01-01

    In physiopathological conditions, such as diabetes, wound healing is significantly compromised and chronic complications, including ulcers, may occur. In a mouse model of skin repair, we recently reported that wound treatment with Sirtuin activators and class I HDAC inhibitors induced keratinocyte proliferation and enhanced healing via a nitric oxide (NO) dependent mechanism. We observed an increase in total protein acetylation in the wound area, as determined by acetylation of α-tubulin and histone H3 Lysine 9. We reasoned that this process activated cell function as well as regulated gene expression to foster tissue repair. We report here that the direct activation of P300/CBP-associated factor (PCAF) by the histone acetylase activator pentadecylidenemalonate 1b (SPV-106) induced Lysine acetylation in the wound area. This intervention was sufficient to enhance repair process by a NO-independent mechanism. Hence, an impairment of PCAF and/or other GCN5 family acetylases may delay skin repair in physiopathological conditions. PMID:24265859

  15. Systematic Analysis of the Lysine Acetylome in Candida albicans.

    PubMed

    Zhou, Xiaowei; Qian, Guanyu; Yi, Xingling; Li, Xiaofang; Liu, Weida

    2016-08-01

    Candida albicans (C. albicans) is a worldwide cause of fungal infectious diseases. As a general post-translational modification (PTM), lysine acetylation of proteins play an important regulatory role in almost every cell. In our research, we used a high-resolution proteomic technique (LC-MS/MS) to present the comprehensive analysis of the acetylome in C. albicans. In general, we detected 477 acetylated proteins among all 9038 proteins (5.28%) in C. albicans, which had 1073 specific acetylated sites. The bioinformatics analysis of the acetylome showed a significant role in the regulation of metabolism. To be more precise, proteins involved in carbon metabolism and biosynthesis were the underlying objectives of acetylation. Besides, through the study of the acetylome, we found a universal rule in acetylated motifs: the +4, +5, or +6 position, which is an alkaline residue with a long side chain (K or R), and the +1 or +2 position, which is a residue with a long side chain (Y, H, W, or F). To the best of our knowledge, all screening acetylated histone sites of this study have not been previously reported. Moreover, protein-protein interaction network (PPI) study demonstrated that a variety of connections in glycolysis/gluconeogenesis, oxidative phosphorylation, and the ribosome were modulated by acetylation and phosphorylation, but the phosphorylated proteins in oxidative phosphorylation PPI network were not abundant, which indicated that acetylation may have a more significant effect than phosphorylation on oxidative phosphorylation. This is the first study of the acetylome in human pathogenic fungi, providing an important starting point for the in-depth discovery of the functional analysis of acetylated proteins in such fungal pathogens. PMID:27297460

  16. The kinetics of hydrolysis of some extended N-aminoacyl-l-lysine methyl esters.

    PubMed

    Green, G D; Tomalin, G

    1976-09-01

    1. The action of two active forms of bovine trypsin (alpha and beta-trypsin) on a series of specific methyl ester substrates of general formula: N-acetyl-(glycyl)n-L-lysine methyl ester (n = 0, 1, 2) and N2-benzoyl-L-arginine ethyl ester have been investigated. With the L-lysine methyl esters the catalytic rate constant for hydrolysis (kcat) was found to be significantly lower for alpha-trypsin than for beta-trypsin, whereas with N2-benzoyl-L-arginine ethyl ester there was no significant difference for the two enzymes. 2. By measurement of the kinetic constants (kcat and Km) in the presence of a nucleophile, which competes with water in the deacylation process, it has been shown that, in common with the specific ester substrates of trypsin, the rate-determining step for the extended L-lysine methyl esters is decaylation of the enzyme. 3. It has been found that by extending the aminoacyl group of N-acetyl-L-lysine methyl ester by one glycine residue (n = 1), a greatly enhanced deacylation rate constant is observed for both alpha and beta-trypsin. The higher rate constants were maintained at the higher levels by the addition of a further glycine residue (n = 2). These results have been interpreted in terms of the 'induced fit' hypothesis the substrates binding to an enzyme subsite adjacent to the active site. 4. The beta-trypsin-catalysed hydrolysis of the L-lysine substrates was investigated over a range of temperature (15--35 degrees C). The Arrhenius law was obeyed, within experimental error, by all three substrates allowing the estimation of the thermodynamic function of activation (delta S not equal to and deltaH note equal to) for the deacylation reactions. The significantly higher values of deltaS not equal to and deltaH not equal to obtained for the two extended substrates are interpreted in terms of additional hydrogen bonding between the longer aminoacyl chains and the enzyme molecule. The results are compared with those for non-extended specific substrates

  17. System-wide Studies of N-Lysine Acetylation in Rhodopseudomonas palustris Reveal Substrate Specificity of Protein Acetyltransferases*

    PubMed Central

    Crosby, Heidi A.; Pelletier, Dale A.; Hurst, Gregory B.; Escalante-Semerena, Jorge C.

    2012-01-01

    N-Lysine acetylation is a posttranslational modification that has been well studied in eukaryotes and is likely widespread in prokaryotes as well. The central metabolic enzyme acetyl-CoA synthetase is regulated in both bacteria and eukaryotes by acetylation of a conserved lysine residue in the active site. In the purple photosynthetic α-proteobacterium Rhodopseudomonas palustris, two protein acetyltransferases (RpPat and the newly identified RpKatA) and two deacetylases (RpLdaA and RpSrtN) regulate the activities of AMP-forming acyl-CoA synthetases. In this work, we used LC/MS/MS to identify other proteins regulated by the N-lysine acetylation/deacetylation system of this bacterium. Of the 24 putative acetylated proteins identified, 14 were identified more often in a strain lacking both deacetylases. Nine of these proteins were members of the AMP-forming acyl-CoA synthetase family. RpPat acetylated all nine of the acyl-CoA synthetases identified by this work, and RpLdaA deacetylated eight of them. In all cases, acetylation occurred at the conserved lysine residue in the active site, and acetylation decreased activity of the enzymes by >70%. Our results show that many different AMP-forming acyl-CoA synthetases are regulated by N-lysine acetylation. Five non-acyl-CoA synthetases were identified as possibly acetylated, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Rpa1177, a putative 4-oxalocrotonate tautomerase. Neither RpPat nor RpKatA acetylated either of these proteins in vitro. It has been reported that Salmonella enterica Pat (SePat) can acetylate a number of metabolic enzymes, including GAPDH, but we were unable to confirm this claim, suggesting that the substrate range of SePat is not as broad as suggested previously. PMID:22416131

  18. Acetylation of Lysine92 Improves the Chaperone and Anti-apoptotic Activities of Human αB-Crystallin

    PubMed Central

    Nahomi, Rooban B.; Huang, Rong; Nandi, Sandip K.; Wang, Benlian; Padmanabha, Smitha; Santhoshkumar, Puttur; Filipek, Slawomir; Biswas, Ashis; Nagaraj, Ram H.

    2013-01-01

    αB-Crystallin is a chaperone and an anti-apoptotic protein that is highly expressed in many tissues, including the lens, retina, heart and kidney. In the human lens, several lysine residues in αB-crystallin are acetylated. We have previously shown that such acetylation is predominant at lysine92 (K92) and K166. We have investigated the effect of lysine acetylation on the structure and functions of αB-crystallin by the specific introduction of an Nε-acetyllysine (AcK) mimic at K92. The introduction of AcK slightly altered the secondary and tertiary structures of the protein. AcK introduction also resulted in an increase in the molar mass and hydrodynamic radius of the protein, and the protein became structurally more open and more stable than the native protein. The acetyl protein acquired higher surface hydrophobicity and exhibited 25-55% higher chaperone activity than the native protein. The acetyl protein had higher client protein binding per subunit of the protein and higher binding affinity relative to the native protein. The acetyl protein was at least 20% more effective in inhibiting chemically induced apoptosis than the native protein. Molecular modeling suggests that acetylation of K92 makes the ‘α-crystallin domain’ more hydrophobic. Together, our results reveal that the acetylation of a single lysine residue in αB-crystallin makes the protein structurally more stable and improves its chaperone and anti-apoptotic activities. Our findings suggest that lysine acetylation of αB-crystallin is an important chemical modification to enhance αB-crystallin’s protective functions in the eye. PMID:24128140

  19. Molecular cloning and characterization of chicken NK lysin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    NK lysin is an anti microbial and anti tumor protein expressed by NK cells and T lymphocytes. In a previous report, we identified a set of overlapping expressed sequence tags constituting a contiguous sequence (contig 171) homologous to mammalian NK lysins. In the current report, a cDNA encoding N...

  20. 40 CFR 721.10250 - Zirconium lysine complex (generic).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Zirconium lysine complex (generic... Specific Chemical Substances § 721.10250 Zirconium lysine complex (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as...

  1. 40 CFR 721.10250 - Zirconium lysine complex (generic).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Zirconium lysine complex (generic... Specific Chemical Substances § 721.10250 Zirconium lysine complex (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as...

  2. 40 CFR 721.10250 - Zirconium lysine complex (generic).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Zirconium lysine complex (generic... Specific Chemical Substances § 721.10250 Zirconium lysine complex (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as...

  3. Identification of the critical linker residues conferring differences in the compactness of NS5 from Dengue virus serotype 4 and NS5 from Dengue virus serotypes 1-3.

    PubMed

    Subramanian Manimekalai, Malathy Sony; Saw, Wuan Geok; Pan, Ankita; Grüber, Ardina; Grüber, Gerhard

    2016-06-01

    Dengue virus (DENV) nonstructural protein 5 (NS5) consists of a methyltransferase (MTase) domain and an RNA-dependent RNA polymerase (RdRp) domain. The cross-talk between these domains occurs via a ten-residue linker. Recent solution studies of DENV NS5 from all four serotypes (DENV-1 to DENV-4) showed that NS5 adopts multiple conformations owing to its flexible linker and that DENV-4 NS5 is more compact and less flexible compared with NS5 from DENV-1 to DENV-3 [Saw et al. (2015), Acta Cryst. D71, 2309-2327]. Here, using a variety of single, double, triple and quadruple mutants of DENV-4 NS5 combined with solution X-ray scattering studies, insight into the critical residues responsible for the differential flexibility of DENV-4 NS5 is presented. The DENV-4 NS5 mutants K271T and S266N/T267A as well as the deletion mutant ΔS266T267 showed enlarged dimensions and flexibility similar to those of DENV-3 NS5. The data indicate that the residues Lys271, Ser266 and Thr267 are important for the compactness of DENV-4 NS5 and therefore may be critical for the regulation of virus replication. Furthermore, quantitative characterization of the flexibility of these DENV-4 NS5 linker mutants using the ensemble-optimization method revealed that these mutants possess a similar conformational distribution to DENV-3 NS5, confirming that these residues in the linker region cause the higher compactness of DENV-4 NS5. PMID:27303800

  4. Probing the Role of Two Critical Residues in Inulin Fructotransferase (DFA III-Producing) Thermostability from Arthrobacter sp. 161MFSha2.1.

    PubMed

    Yu, Shuhuai; Wang, Xiao; Zhang, Tao; Jiang, Bo; Mu, Wanmeng

    2016-08-10

    Inulin fructotransferase (IFTase) is an important enzyme that produces di-d-fructofuranose 1,2':2,3' dianhydride (DAF III), which is beneficial for human health. Present investigations mainly focus on screening and characterizing IFTase, including catalytic efficiency and thermostability, which are two important factors for enzymatic industrial applications. However, few reports aimed to improve these two characteristics based on the structure of IFTase. In this work, a structural model of IFTase (DFA III-producing) from Arthrobacter sp. 161MFSha2.1 was constructed through homology modeling. Analysis of this model reveals that two residues, Ser-309 and Ser-333, may play key roles in the structural stability. Therefore, the functions of the two residues were probed by site-directed mutagenesis combined with the Nano-DSC method and assays for residual activity. In contrast to other mutations, single mutation of serine 309 (or serine 333) to threonine did not decrease the enzymatic stability, whereas double mutation (serine 309 and serine 333 to threonine) can enhance thermostability (by approximately 5 °C). The probable mechanisms for this enhancement were investigated. PMID:27440442

  5. Quantification of Nε-(2-Furoylmethyl)-L-lysine (furosine), Nε-(Carboxymethyl)-L-lysine (CML), Nε-(Carboxyethyl)-L-lysine (CEL) and total lysine through stable isotope dilution assay and tandem mass spectrometry.

    PubMed

    Troise, Antonio Dario; Fiore, Alberto; Wiltafsky, Markus; Fogliano, Vincenzo

    2015-12-01

    The control of Maillard reaction (MR) is a key point to ensure processed foods quality. Due to the presence of a primary amino group on its side chain, lysine is particularly prone to chemical modifications with the formation of Amadori products (AP), Nε-(Carboxymethyl)-L-lysine (CML), Nε-(Carboxyethyl)-L-lysine (CEL). A new analytical strategy was proposed which allowed to simultaneously quantify lysine, CML, CEL and the Nε-(2-Furoylmethyl)-L-lysine (furosine), the indirect marker of AP. The procedure is based on stable isotope dilution assay followed by liquid chromatography tandem mass spectrometry. It showed high sensitivity and good reproducibility and repeatability in different foods. The limit of detection and the RSD% were lower than 5 ppb and below 8%, respectively. Results obtained with the new procedure not only improved the knowledge about the reliability of thermal treatment markers, but also defined new insights in the relationship between Maillard reaction products and their precursors. PMID:26041204

  6. Lysine catabolism in Rhizoctonia leguminicola and related fungi.

    PubMed Central

    Guengerich, F P; Broquist, H P

    1976-01-01

    The catabolism of lysine was studied in several yeasts and fungi. Results with cell-free extracts of Rhizoctonia leguminicola support a proposed pathway involving (D- and L-) EPSILON-N-acetyllysine, alpha-keto-epsilon-acetamidohexanoic acid, delta-acetamidovaleric acid, and delta-aminovaleric acid in the conversion of L-lysine to shortchain organic acids. Label from radioactive L-lysine was found to accumulate in D- and L-epsilon-N-acetyllysine, delta-acetamidovaleric acid, delta-aminovaleric acid, and glutaric acid in cultures of R. leguminicola, Neurospora crassa, Saccharomyces cerevisiae, and Hansenula saturnus, suggesting that the proposed omega-acetyl pathway of lysine catabolism is generalized among yeasts and fungi. In N. crassa, as is the case in R. leguminicola, the major precursor of L-pipecolic acid was the L-isomer of lysine; 15N experiments were consistent with delta1-piperideine-2-carboxylic acid as an intermediate in the transformation. PMID:131119

  7. Role of several histone lysine methyltransferases in tumor development

    PubMed Central

    LI, JIFU; ZHU, SHUNQIN; KE, XIAO-XUE; CUI, HONGJUAN

    2016-01-01

    The field of cancer epigenetics has been evolving rapidly in recent decades. Epigenetic mechanisms include DNA methylation, histone modifications and microRNAs. Histone modifications are important markers of function and chromatin state. Aberrant histone methylation frequently occurs in tumor development and progression. Multiple studies have identified that histone lysine methyltransferases regulate gene transcription through the methylation of histone, which affects cell proliferation and differentiation, cell migration and invasion, and other biological characteristics. Histones have variant lysine sites for different levels of methylation, catalyzed by different lysine methyltransferases, which have numerous effects on human cancers. The present review focused on the most recent advances, described the key function sites of histone lysine methyltransferases, integrated significant quantities of data to introduce several compelling histone lysine methyltransferases in various types of human cancers, summarized their role in tumor development and discussed their potential mechanisms of action. PMID:26998265

  8. CE-LIF determination of salivary cadaverine and lysine concentration ratio as an indicator of lysine decarboxylase enzyme activity.

    PubMed

    Tábi, Tamás; Lohinai, Zsolt; Pálfi, Melinda; Levine, Martin; Szöko, Eva

    2008-05-01

    Salivary bacteria produce the enzyme lysine decarboxylase which converts lysine to cadaverine. In the absence of appropriate oral hygiene, overgrowth of these bacteria depletes lysine. This may contribute to gingival inflammation, while cadaverine contributes to oral malodor. A selective and sensitive capillary electrophoresis method with laser-induced fluorescence detection has been developed for the determination of cadaverine and lysine in saliva, as an indicator of lysine decarboxylase enzyme activity. The diamino compounds were separated in acidic background electrolyte in their mono-labeled form after derivatization with 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole (NBD-F). Linearity and reproducibility of the method in the range 1-50 μmol L(-1) have been demonstrated using saliva samples. The method was applied for the measurement of cadaverine and lysine in the saliva of healthy volunteers with or without proper oral hygiene. In the absence of oral hygiene, the mol fraction of cadaverine to cadaverine plus lysine in saliva increased significantly (0.65 ± 0.13 vs. 0.39 ± 0.18, P < 0.001), indicating the presence of higher amount of bacterial lysine decarboxylase, that may contribute to periodontal diseases. PMID:18389226

  9. Chiral assemblies of nickel lysinate via the corrosive adsorption of (S)-lysine on Ni/Au{111}

    NASA Astrophysics Data System (ADS)

    Wilson, K. E.; Baddeley, C. J.

    2014-11-01

    The adsorption of (S)-lysine onto submonolayer coverages of Ni on Au{111} was investigated by scanning tunnelling microscopy and reflection absorption infrared spectroscopy. Arrays of two-dimensional Ni nanoclusters were prepared on the Au{111} surface. The sticking probability of (S)-lysine was found to increase by an order of magnitude on Au surfaces templated by Ni compared to the clean Au surface. (S)-lysine corrodes Ni from the edges of clusters forming nickel lysinate complexes which self-assemble to form ordered molecular arrays. Below a threshold coverage, the Ni clusters are completely destroyed by (S)-lysine adsorption. Under these conditions, extensive restructuring of the Au steps is observed. The implications of our work for understanding the role of chiral modifiers in Ni catalysed enantioselective catalysis are discussed.

  10. Evolution of metal-compound residues on the walls of plasma etching reactor and their effect on critical dimensions of high-k/metal gate

    SciTech Connect

    Iwakoshi, Takehisa; Ono, Tetsuo; Aoyama, Takayuki; Nara, Yasuo; Ohji, Yuzuru

    2009-05-15

    It was found that critical dimensions of high-k/metal gates obey the multivariate linear approximation with the precision of 3{sigma}={+-}0.86 nm, whose explanatory variables are amounts of metal compounds remaining on the plasma reactor walls. To measure their amounts, the authors assumed they are proportional to amounts of atoms sputtered out by Ar plasma and falling onto a Si wafers placed on a wafer stage. In this study, effects of metal compounds of W, Ti, Ta, and Hf, which are used to construct full-metal/high-k gates, were measured. It was found that Ti and Ta compounds dominate the fluctuation of critical dimensions and the dependency of their amount on wafer numbers being etched obeys a simple difference equation. From these results, they can estimate and minimize the fluctuations of critical dimensions in mass fabrications.

  11. Structure-function relationship of a plant NCS1 member--homology modeling and mutagenesis identified residues critical for substrate specificity of PLUTO, a nucleobase transporter from Arabidopsis.

    PubMed

    Witz, Sandra; Panwar, Pankaj; Schober, Markus; Deppe, Johannes; Pasha, Farhan Ahmad; Lemieux, M Joanne; Möhlmann, Torsten

    2014-01-01

    Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members. PMID:24621654

  12. Structure-Function Relationship of a Plant NCS1 Member – Homology Modeling and Mutagenesis Identified Residues Critical for Substrate Specificity of PLUTO, a Nucleobase Transporter from Arabidopsis

    PubMed Central

    Witz, Sandra; Panwar, Pankaj; Schober, Markus; Deppe, Johannes; Pasha, Farhan Ahmad; Lemieux, M. Joanne; Möhlmann, Torsten

    2014-01-01

    Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members. PMID:24621654

  13. Solubility Behavior of Cyanophycin Depending on Lysine Content

    PubMed Central

    Wiefel, Lars

    2014-01-01

    Study of the synthesis of cyanophycin (CGP) in recombinant organisms focused for a long time mostly on the insoluble form of CGP, due to its easy purification and its putative use as a precursor for biodegradable chemicals. Recently, another form of CGP, which, in contrast to the insoluble form, was soluble at neutral pH, became interesting due to its high lysine content, which was also assumed to be the reason for the solubility of the polymer. In this study, we demonstrate that lysine incorporated into insoluble CGP affected the solubility of the polymer in relation to its lysine content. Insoluble CGP can be separated along a temperature gradient of 90°C to 30°C, where CGP showed an increasing lysine content corresponding to a decreasing temperature needed for solubilization. CGP with less than 3 to 4 mol% lysine did not become soluble even at 90°C, while CGP with 31 mol% lysine was soluble at 30°C. In lysine fractions at higher than 31 mol%, CGP was soluble. The temperature separation will be suitable for improving the downstream processing of CGP synthesized in large-scale fermentations, including faster and more efficient purification of CGP, as well as enrichment and separation of dipeptides and CGP with specific amino acid compositions. PMID:24271185

  14. Bacteriocin AS-48, a microbial cyclic polypeptide structurally and functionally related to mammalian NK-lysin

    PubMed Central

    González, Carlos; Langdon, Grant M.; Bruix, Marta; Gálvez, Antonio; Valdivia, Eva; Maqueda, Mercedes; Rico, Manuel

    2000-01-01

    The solution structure of bacteriocin AS-48, a 70-residue cyclic polypeptide from Enterococcus faecalis, consists of a globular arrangement of five α-helices enclosing a compact hydrophobic core. The head-to-tail union lies in the middle of helix 5, a fact that is shown to have a pronounced effect on the stability of the three-dimensional structure. Positive charges in the side chains of residues in helix 4 and in the turn linking helix 4 to helix 5 form a cluster that most probably determine its antibacterial activity by promoting pore formation in cell membranes. A similar five-helix structural motif has been found in the antimicrobial NK-lysin, an effector polypeptide of T and natural killer (NK) cells. Bacteriocin AS-48 lacks the three disulfide bridges characteristic of the saposin fold present in NK-lysin, and has no sequence homology with it. Nevertheless, the similar molecular architecture and high positive charge strongly suggest a common mechanism of antibacterial action. PMID:11005847

  15. Structural basis for G9a-like protein lysine methyltransferase inhibition by BIX-01294

    SciTech Connect

    Chang, Yanqi; Zhang, Xing; Horton, John R.; Upadhyay, Anup K.; Spannhoff, Astrid; Liu, Jin; Synder, James P.; Bedford, Mark T.; Cheng, Xiaodong

    2009-03-26

    Histone lysine methylation is an important epigenetic mark that regulates gene expression and chromatin organization. G9a and G9a-like protein (GLP) are euchromatin-associated methyltransferases that repress transcription by methylating histone H3 Lys9. BIX-01294 was originally identified as a G9a inhibitor during a chemical library screen of small molecules and has previously been used in the generation of induced pluripotent stem cells. Here we present the crystal structure of the catalytic SET domain of GLP in complex with BIX-01294 and S-adenosyl-L-homocysteine. The inhibitor is bound in the substrate peptide groove at the location where the histone H3 residues N-terminal to the target lysine lie in the previously solved structure of the complex with histone peptide. The inhibitor resembles the bound conformation of histone H3 Lys4 to Arg8, and is positioned in place by residues specific for G9a and GLP through specific interactions.

  16. Lysine mediation of neuroendocrine food regulation in guinea fowl.

    PubMed

    Payne, A; Wang, X; Ivy, M T; Stewart, A; Nelson, K; Darris, C; Nahashon, S N

    2016-02-01

    In poultry, obesity is partly influenced by food intake, and is increasingly becoming a nationwide problem. Hypothalamic food intake mechanisms are involved metabolically and neurologically via two peptide hormones, leptin and ghrelin, and the amino acid glutamate, which is enzymatically derived from lysine metabolism. We hypothesize that lysine homeostasis mediates regulation of feed intake and performance characteristics via the brain-liver axis through glutamate sensing. The objective was to examine the effects of lysine homeostasis in avian food regulation and performance through neuroendocrine signaling. One-day-old male French Guinea fowl (GF) keets (n = 270) were weighed and randomly assigned to 5 dietary treatments (0.80%, 0.86%, 0.92%, 1.10% control, and 1.22% lysine) in 3 replicates. At 4 and 8 wk of age 20% of experimental birds were randomly selected, weighed and euthanatized. The liver, pancreas, and hypothalamus were excised, snap frozen in liquid nitrogen and stored at -80°C until use. Tissue mRNA was extracted and cDNA synthesized for qPCR assays. Lysine at 0.80 and 0.86% hindered growth, development of digestive organs, expression of brain and liver glutamate and leptin receptors, and caused high mortality in GF. The fold change for metabotropic glutamate receptor I was lower (P < 0.05) in liver and higher in brain at 0.86 and 0.92% than the control (1.10%) and 1.22% lysine. The 1.22% lysine exhibited highest expression of ionotropic glutamate receptor, while brain ghrelin receptor expression was highest at 0.86 and 0.92% lysine. Therefore, dietary lysine concentration may influence signaling pathways regulating food intake in brain-liver axis via glutamate synthesis. PMID:26614682

  17. Identification of N(α)-acetyl-α-lysine as a probable thermolyte and its accumulation mechanism in Salinicoccus halodurans H3B36.

    PubMed

    Jiang, Kai; Xue, Yanfen; Ma, Yanhe

    2015-01-01

    Salinicoccus halodurans H3B36 is a moderate halophile that was isolated from a 3.2-m-deep sediment sample in Qaidam Basin, China. Our results suggest that N(α)-acetyl-α-lysine can accumulate and act as a probable thermolyte in this strain. The accumulation mechanism and biosynthetic pathway for this rare compatible solute were also elucidated. We confirmed that the de novo synthesis pathway of N(α)-acetyl-α-lysine in this strain starts from aspartate and passes through lysine. Through RNA sequencing, we also found an 8-gene cluster (orf_1582-1589) and another gene (orf_2472) that might encode the biosynthesis of N(α)-acetyl-α-lysine in S. halodurans H3B36. Orf_192, orf_193, and orf_1259 might participate in the transportation of precursors for generating N(α)-acetyl-α-lysine under the heat stress. The transcriptome reported here also generated a global view of heat-induced changes and yielded clues for studying the regulation of N(α)-acetyl-α-lysine accumulation. Heat stress triggered a global transcriptional disturbance and generated a series of actions to adapt the strain to heat stress. Furthermore, the transcriptomic results showed that the regulon of RpoN (orf_2534) may be critical to conferring heat stress tolerance and survival to S. halodurans. PMID:26687465

  18. Identification of Nα-acetyl-α-lysine as a probable thermolyte and its accumulation mechanism in Salinicoccus halodurans H3B36

    PubMed Central

    Jiang, Kai; Xue, Yanfen; Ma, Yanhe

    2015-01-01

    Salinicoccus halodurans H3B36 is a moderate halophile that was isolated from a 3.2-m-deep sediment sample in Qaidam Basin, China. Our results suggest that Nα-acetyl-α-lysine can accumulate and act as a probable thermolyte in this strain. The accumulation mechanism and biosynthetic pathway for this rare compatible solute were also elucidated. We confirmed that the de novo synthesis pathway of Nα-acetyl-α-lysine in this strain starts from aspartate and passes through lysine. Through RNA sequencing, we also found an 8-gene cluster (orf_1582–1589) and another gene (orf_2472) that might encode the biosynthesis of Nα-acetyl-α-lysine in S. halodurans H3B36. Orf_192, orf_193, and orf_1259 might participate in the transportation of precursors for generating Nα-acetyl-α-lysine under the heat stress. The transcriptome reported here also generated a global view of heat-induced changes and yielded clues for studying the regulation of Nα-acetyl-α-lysine accumulation. Heat stress triggered a global transcriptional disturbance and generated a series of actions to adapt the strain to heat stress. Furthermore, the transcriptomic results showed that the regulon of RpoN (orf_2534) may be critical to conferring heat stress tolerance and survival to S. halodurans. PMID:26687465

  19. Dissecting the role of critical residues and substrate preference of a Fatty Acyl-CoA Synthetase (FadD13) of Mycobacterium tuberculosis.

    PubMed

    Khare, Garima; Gupta, Vibha; Gupta, Rakesh K; Gupta, Radhika; Bhat, Rajiv; Tyagi, Anil K

    2009-01-01

    Newly emerging multi-drug resistant strains of Mycobacterium tuberculosis (M.tb) severely limit the treatment options for tuberculosis (TB); hence, new antitubercular drugs are urgently needed. The mymA operon is essential for the virulence and intracellular survival of M.tb and thus represents an attractive target for the development of new antitubercular drugs. This study is focused on the structure-function relationship of Fatty Acyl-CoA Synthetase (FadD13, Rv3089) belonging to the mymA operon. Eight site-directed mutants of FadD13 were designed, constructed and analyzed for the structural-functional integrity of the enzyme. The study revealed that mutation of Lys(487) resulted in approximately 95% loss of the activity thus demonstrating its crucial requirement for the enzymatic activity. Comparison of the kinetic parameters showed the residues Lys(172) and Ala(302) to be involved in the binding of ATP and Ser(404) in the binding of CoenzymeA. The influence of mutations of the residues Val(209) and Trp(377) emphasized their importance in maintaining the structural integrity of FadD13. Besides, we show a synergistic influence of fatty acid and ATP binding on the conformation and rigidity of FadD13. FadD13 represents the first Fatty Acyl-CoA Synthetase to display biphasic kinetics for fatty acids. FadD13 exhibits a distinct preference for C(26)/C(24) fatty acids, which in the light of earlier reported observations further substantiates the role of the mymA operon in remodeling the cell envelope of intracellular M.tb under acidic conditions. A three-dimensional model of FadD13 was generated; the docking of ATP to the active site verified its interaction with Lys(172), Ala(302) and Lys(487) and corresponded well with the results of the mutational studies. Our study provides a significant understanding of the FadD13 protein including the identification of residues important for its activity as well as in the maintenance of structural integrity. We believe that the

  20. Examining the Impact of Gene Variants on Histone Lysine Methylation

    PubMed Central

    Van Rechem, Capucine; Whetstine, Johnathan R.

    2015-01-01

    In recent years, there has been a boom in the amount of genome-wide sequencing data that has uncovered important and unappreciated links between certain genes, families of genes and enzymatic processes and diseases such as cancer. Such studies have highlighted the impact that chromatin modifying enzymes could have in cancer and other genetic diseases. In this review, we summarize characterized mutations and single nucleotide polymorphisms (SNPs) in histone lysine methyltransferases (KMTs), histone lysine demethylases (KDMs) and histones. We primarily focus on variants with strong disease correlations and discuss how they could impact histone lysine methylation dynamics and gene regulation. PMID:24859469

  1. Mutants of Saccharomycopsis lipolytica defective in lysine catabolism.

    PubMed Central

    Gaillardin, C; Fournier, P; Sylvestre, G; Heslot, H

    1976-01-01

    Wild-type strains of Saccharomycopsis lipolytica are able to use lysine as a carbon or a nitrogen source, but not as a unique source for both. Mutants were selected that could not use lysine either as a nitrogen or as a carbon source. Some of them, however, utilized N-6-acetyllysine or 5-aminovaleric acid. Many of the mutants appeared to be blocked in both utilizations, suggesting a unique pathway for lysine degradation (either as a carbon or as a nitrogen source). Genetic characterization of these mutants was achieved by complementation and recombination tests. PMID:1245461

  2. Succinylome Analysis Reveals the Involvement of Lysine Succinylation in Metabolism in Pathogenic Mycobacterium tuberculosis*

    PubMed Central

    Yang, Mingkun; Wang, Yan; Chen, Ying; Cheng, Zhongyi; Gu, Jing; Deng, Jiaoyu; Bi, Lijun; Chen, Chuangbin; Mo, Ran; Wang, Xude; Ge, Feng

    2015-01-01

    Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, remains one of the most prevalent human pathogens and a major cause of mortality worldwide. Metabolic network is a central mediator and defining feature of the pathogenicity of Mtb. Increasing evidence suggests that lysine succinylation dynamically regulates enzymes in carbon metabolism in both bacteria and human cells; however, its extent and function in Mtb remain unexplored. Here, we performed a global succinylome analysis of the virulent Mtb strain H37Rv by using high accuracy nano-LC-MS/MS in combination with the enrichment of succinylated peptides from digested cell lysates and subsequent peptide identification. In total, 1545 lysine succinylation sites on 626 proteins were identified in this pathogen. The identified succinylated proteins are involved in various biological processes and a large proportion of the succinylation sites are present on proteins in the central metabolism pathway. Site-specific mutations showed that succinylation is a negative regulatory modification on the enzymatic activity of acetyl-CoA synthetase. Molecular dynamics simulations demonstrated that succinylation affects the conformational stability of acetyl-CoA synthetase, which is critical for its enzymatic activity. Further functional studies showed that CobB, a sirtuin-like deacetylase in Mtb, functions as a desuccinylase of acetyl-CoA synthetase in in vitro assays. Together, our findings reveal widespread roles for lysine succinylation in regulating metabolism and diverse processes in Mtb. Our data provide a rich resource for functional analyses of lysine succinylation and facilitate the dissection of metabolic networks in this life-threatening pathogen. PMID:25605462

  3. X-ray analysis of residual stress gradients in TiN coatings by a Laplace space approach and cross-sectional nanodiffraction: a critical comparison

    PubMed Central

    Stefenelli, Mario; Todt, Juraj; Riedl, Angelika; Ecker, Werner; Müller, Thomas; Daniel, Rostislav; Burghammer, Manfred; Keckes, Jozef

    2013-01-01

    Novel scanning synchrotron cross-sectional nanobeam and conventional laboratory as well as synchrotron Laplace X-ray diffraction methods are used to characterize residual stresses in exemplary 11.5 µm-thick TiN coatings. Both real and Laplace space approaches reveal a homogeneous tensile stress state and a very pronounced compressive stress gradient in as-deposited and blasted coatings, respectively. The unique capabilities of the cross-sectional approach operating with a beam size of 100 nm in diameter allow the analysis of stress variation with sub-micrometre resolution at arbitrary depths and the correlation of the stress evolution with the local coating microstructure. Finally, advantages and disadvantages of both approaches are extensively discussed. PMID:24068842

  4. HDAC inhibitors induce global changes in histone lysine and arginine methylation and alter expression of lysine demethylases.

    PubMed

    Lillico, Ryan; Sobral, Marina Gomez; Stesco, Nicholas; Lakowski, Ted M

    2016-02-01

    Histone deacetylase (HDAC) inhibitors are cancer treatments that inhibit the removal of the epigenetic modification acetyllysine on histones, resulting in altered gene expression. Such changes in expression may influence other histone epigenetic modifications. We describe a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify lysine acetylation and methylation and arginine methylation on histones extracted from cultured cells treated with HDAC inhibitors. The HDAC inhibitors vorinostat, mocetinostat and entinostat induced 400-600% hyperacetylation in HEK 293 and K562 cells. All HDAC inhibitors decreased histone methylarginines in HEK 293 cells but entinostat produced dose dependent reductions in asymmetric dimethylarginine, not observed in K562 cells. Vorinostat produced increases in histone lysine methylation and decreased expression of some lysine demethylases (KDM), measured by quantitative PCR. Entinostat had variable effects on lysine methylation and decreased expression of some KDM while increasing expression of others. Mocetinostat produced dose dependent increases in histone lysine methylation by LC-MS/MS. This was corroborated with a multiplex colorimetric assay showing increases in histone H3 lysine 4, 9, 27, 36 and 79 methylation. Increases in lysine methylation were correlated with dose dependent decreases in the expression of seven KDM. Mocetinostat functions as an HDAC inhibitor and a de facto KDM inhibitor. PMID:26721445

  5. Reactions of lysine with montmorillonite at 80 degrees C: implications for optical activity, H+ transfer and lysine-montmorillonite binding.

    PubMed

    Cuadros, Javier; Aldega, Luca; Vetterlein, Jonathan; Drickamer, Kurt; Dubbin, William

    2009-05-01

    Amino acid-smectite interaction may have catalyzed prebiotic reactions essential for the emergence of life. Lysine solutions (0.05 M) were reacted with Na-smectite in adsorption-desorption experiments. The lysine-smectite complexes were heated at 80 degrees C for 10 days to investigate (1) possible slow processes taking place at surface temperature that would be accelerated at higher temperature and (2) processes taking place in hydrothermal systems. Three sets of experiments were performed: thermal treatment in closed tubes and water added regularly; thermal treatment in closed tubes without adding water; and thermal treatment in open tubes and no added water. After lysine desorption (displacement with 0.1 M CaCl(2)), the solutions were investigated using circular dichroism (CD) and the smectite samples using FTIR and CHN elemental analysis. CD spectra were dependent on the solution pH, which was controlled by lysine protonation state. The lysine protonation state was altered by the adsorption-desorption process, with a higher Lys(+)/Lys(+/-) ratio after desorption. The CD and CHN analyses show that the thermal treatment in a moist state causes stronger smectite-lysine binding. FTIR data suggest that the stronger binding is caused by more or stronger H bonds between -NH(3)(+) lysine groups and smectite basal O atoms. PMID:19185874

  6. Metabolic engineering Corynebacterium glutamicum for the L-lysine production by increasing the flux into L-lysine biosynthetic pathway.

    PubMed

    Xu, Jianzhong; Han, Mei; Zhang, Junlan; Guo, Yanfeng; Zhang, Weiguo

    2014-09-01

    The experiments presented here were based on the conclusions of our previous results. In order to avoid introduction of expression plasmid and to balance the NADH/NAD ratio, the NADH biosynthetic enzyme, i.e., NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GADPH), was replaced by NADP-dependent GADPH, which was used to biosynthesize NADPH rather than NADH. The results indicated that the NADH/NAD ratio significantly decreased, and glucose consumption and L-lysine production drastically improved. Moreover, increasing the flux through L-lysine biosynthetic pathway and disruption of ilvN and hom, which involve in the branched amino acid and L-methionine biosynthesis, further improved L-lysine production by Corynebacterium glutamicum. Compared to the original strain C. glutamicum Lys5, the L-lysine production and glucose conversion efficiency (α) were enhanced to 81.0 ± 6.59 mM and 36.45% by the resulting strain C. glutamicum Lys5-8 in shake flask. In addition, the by-products (i.e., L-threonine, L-methionine and L-valine) were significantly decreased as results of genetic modification in homoserine dehydrogenase (HSD) and acetohydroxyacid synthase (AHAS). In fed-batch fermentation, C. glutamicum Lys5-8 began to produce L-lysine at post-exponential growth phase and continuously increased over 36 h to a final titer of 896 ± 33.41 mM. The L-lysine productivity was 2.73 g l(-1) h(-1) and the α was 47.06% after 48 h. However, the attenuation of MurE was not beneficial to increase the L-lysine production because of decreasing the cell growth. Based on the above-mentioned results, we get the following conclusions: cofactor NADPH, precursor, the flux through L-lysine biosynthetic pathway and DCW are beneficial to improve L-lysine production in C. glutamicum. PMID:24879631

  7. NK-lysin, a novel effector peptide of cytotoxic T and NK cells. Structure and cDNA cloning of the porcine form, induction by interleukin 2, antibacterial and antitumour activity.

    PubMed Central

    Andersson, M; Gunne, H; Agerberth, B; Boman, A; Bergman, T; Sillard, R; Jörnvall, H; Mutt, V; Olsson, B; Wigzell, H

    1995-01-01

    A 78 residue antimicrobial, basic peptide, NK-lysin, with three intrachain disulfide bonds was purified from pig small intestine and characterized. A corresponding clone was isolated from a porcine bone marrow cDNA library. The 780 bp DNA sequence had a reading frame of 129 amino acids which corresponded to NK-lysin. The clone was used to show that stimulation with human interleukin-2 induced synthesis of NK-lysin-specific mRNA in a lymphocyte fraction enriched for T and NK cells. Lower levels of mRNA were detected in tissues known to contain T and NK cells, such as small intestine, spleen and colon. Interleukin-2 also induced both proliferation of the lymphocyte fraction and cytolytic function in these cells. Immunostaining showed that NK-lysin was present in cells positive for CD8, CD2 and CD4. NK-lysin showed high anti-bacterial activity against Escherichia coli and Bacillus megaterium and moderate activity against Acinetobacter calcoaceticus and Streptococcus pyogenes. The peptide showed a marked lytic activity against an NK-sensitive mouse tumour cell line, YAC-1, but it did not lyse red blood cells. The amino acid sequence of NK-lysin exhibits 33% identity with a putative human preproprotein, NKG5, of unknown function but derived from a cDNA clone of activated NK cells. We suggest that NK-lysin is a new effector molecule of cytotoxic T and NK cells. Images PMID:7737114

  8. Synthesis and biological activity of a lysine-containing cyclic analog of (Leu/sup 5/)enkephalin

    SciTech Connect

    Bobrova, I.V.; Abissova, N.A.; Rozental', G.F.; Nikiforovich, G.V.; Chipens, G.I.

    1986-09-01

    A cyclic analog of enkephalin - cyclo(Lys-Tyr-Gly-Gly-Phe-Leu) -- and two corresponding linear hexapeptides containing a residue of the amino acid lysine at the beginning and the end of the molecule - Lys-Tyr-Gly-Gly-Phe-Leu and Tyr-Gly-Gly-Phe-Leu-Lys - have been synthesized by the classical methods of peptide chemistry. The addition of a lysine residue to the N-end of the enkephalin molecule or the cyclization of this hexapeptide decreased the action of the analogs on the central and peripheral opiate receptors. The addition of lysine through the epsilon-amino group to the C-end of the enkephalin molecule scarcely changed the interaction of the analog with the ..mu..-type of opiate receptor but lowered its affinity for the delta-type of receptor approximately 10-fold. All three analogs that were synthesized possessed an analgesic activity comparable in magnitude with the activity of (Leu/sup 5/)enkephalin determined by the tail pinch method on intracisternal administration to mice.

  9. Starvation induced alterations in hepatic lysine metabolism in different families of rainbow trout (Oncorhynchus mykiss)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lysine is the second limiting amino acid in fish meal based diets, second only to methionine. However, little is known about lysine metabolism in rainbow trout (RBT). Therefore, lysine catabolism by the lysine alpha-ketoglutarate reductase (LKR) pathway was studied. Additionally, since genetically i...

  10. Crystal structures of SIRT3 reveal that the α2-α3 loop and α3-helix affect the interaction with long-chain acyl lysine.

    PubMed

    Gai, Wei; Li, He; Jiang, Hualiang; Long, Yaqiu; Liu, Dongxiang

    2016-09-01

    SIRT1-7 play important roles in many biological processes and age-related diseases. In addition to a NAD(+) -dependent deacetylase activity, they can catalyze several other reactions, including the hydrolysis of long-chain fatty acyl lysine. To study the binding modes of sirtuins to long-chain acyl lysines, we solved the crystal structures of SIRT3 bound to either a H3K9-myristoylated- or a H3K9-palmitoylated peptide. Interaction of SIRT3 with the palmitoyl group led to unfolding of the α3-helix. The myristoyl and palmitoyl groups bind to the C-pocket and an allosteric site near the α3-helix, respectively. We found that the residues preceding the α3-helix determine the size of the C-pocket. The flexibility of the α2-α3 loop and the plasticity of the α3-helix affect the interaction with long-chain acyl lysine. PMID:27501476

  11. Gramicidin A disassembles large conductive clusters of its lysine-substituted derivatives in lipid membranes.

    PubMed

    Antonenko, Yuri N; Gluhov, Grigory S; Firsov, Alexander M; Pogozheva, Irina D; Kovalchuk, Sergey I; Pechnikova, Evgeniya V; Kotova, Elena A; Sokolova, Olga S

    2015-07-14

    N-terminally substituted lysine derivatives of gramicidin A (gA), [Lys1]gA and [Lys3]gA, but not glutamate- or aspartate-substituted peptides have been previously shown to cause the leakage of carboxyfluorescein from liposomes. Here, the leakage induction was also observed for [Arg1]gA and [Arg3]gA, while [His1]gA and [His3]gA were inactive at neutral pH. The Lys3-containing analogue with all tryptophans replaced by isoleucines did not induce liposome leakage, similar to gA. This suggests that the presence of both tryptophans and N-terminal cationic residues is critical for pore formation. Remarkably, the addition of gA blocked the leakage induced by [Lys3]gA. By examining with fluorescence correlation spectroscopy the peptide-induced leakage of fluorescent markers from liposomes, we estimated the diameter of pores responsible for the leakage to be about 1.6 nm. Transmission electron cryo-microscopy imaging of liposomes with [Lys3]gA showed that the liposomal membranes contained high electron density particles with a size of about 40 Å, suggesting the formation of peptide clusters. No such clusterization was observed in liposomes incorporating gA or a mixture of gA with [Lys3]gA. Three-dimensional reconstruction of the clusters was compatible with their pentameric arrangement. Based on experimental data and computational modeling, we suggest that the large pore formed by [Lys3]gA represents a barrel-stave oligomeric cluster formed by antiparallel double-stranded helical dimers (DH). In a tentative model, the pentamer of dimers may be stabilized by aromatic Trp-Trp and cation-π Trp-Lys interactions between the neighboring DHs. The inhibiting effect of gA on the [Lys3]gA-induced leakage can be attributed to breaking of cation-π interactions, which prevents peptide clusterization and pore formation. PMID:26077982

  12. Data detailing the platelet acetyl-lysine proteome

    PubMed Central

    Aslan, Joseph E.; David, Larry L.; McCarty, Owen J.T.

    2015-01-01

    Here we detail proteomics data that describe the acetyl-lysine proteome of blood platelets (Aslan et al., 2015 [1]). An affinity purification – mass spectrometry (AP-MS) approach was used to identify proteins modified by Nε-lysine acetylation in quiescent, washed human platelets. The data provide insights into potential regulatory mechanisms of platelet function mediated by protein lysine acetylation. Additionally, as platelets are anucleate and lack histone proteins, they offer a unique and valuable system to study the regulation of cytosolic proteins by lysine acetylation. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (Vizcaino et al., 2014 [2]) via with PRIDE partner repository with the dataset identifier PXD002332. PMID:26904711

  13. Data detailing the platelet acetyl-lysine proteome.

    PubMed

    Aslan, Joseph E; David, Larry L; McCarty, Owen J T

    2015-12-01

    Here we detail proteomics data that describe the acetyl-lysine proteome of blood platelets (Aslan et al., 2015 [1]). An affinity purification - mass spectrometry (AP-MS) approach was used to identify proteins modified by Nε-lysine acetylation in quiescent, washed human platelets. The data provide insights into potential regulatory mechanisms of platelet function mediated by protein lysine acetylation. Additionally, as platelets are anucleate and lack histone proteins, they offer a unique and valuable system to study the regulation of cytosolic proteins by lysine acetylation. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (Vizcaino et al., 2014 [2]) via with PRIDE partner repository with the dataset identifier PXD002332. PMID:26904711

  14. Lysine fatty acylation promotes lysosomal targeting of TNF-α

    PubMed Central

    Jiang, Hong; Zhang, Xiaoyu; Lin, Hening

    2016-01-01

    Tumor necrosis factor-α (TNF-α) is a proinflammation cytokine secreted by various cells. Understanding its secretive pathway is important to understand the biological functions of TNF-α and diseases associated with TNF-α. TNF-α is one of the first proteins known be modified by lysine fatty acylation (e.g. myristoylation). We previously demonstrated that SIRT6, a member of the mammalian sirtuin family of enzymes, can remove the fatty acyl modification on TNF-α and promote its secretion. However, the mechanistic details about how lysine fatty acylation regulates TNF-α secretion have been unknown. Here we present experimental data supporting that lysine fatty acylation promotes lysosomal targeting of TNF-α. The result is an important first step toward understanding the biological functions of lysine fatty acylation. PMID:27079798

  15. Stabilization of collagen nanofibers with l-lysine improves the ability of carbodiimide cross-linked amniotic membranes to preserve limbal epithelial progenitor cells

    PubMed Central

    Lai, Jui-Yang; Wang, Pei-Ran; Luo, Li-Jyuan; Chen, Si-Tan

    2014-01-01

    To overcome the drawbacks associated with limited cross-linking efficiency of carbodiimide modified amniotic membrane, this study investigated the use of l-lysine as an additional amino acid bridge to enhance the stability of a nanofibrous tissue matrix for a limbal epithelial cell culture platform. Results of ninhydrin assays and zeta potential measurements showed that the amount of positively charged amino acid residues incorporated into the tissue collagen chains is highly correlated with the l-lysine-pretreated concentration. The cross-linked structure and hydrophilicity of amniotic membrane scaffolding materials affected by the lysine molecular bridging effects were determined. With an increase in the l-lysine-pretreated concentration from 1 to 30 mM, the cross-linking density was significantly increased and water content was markedly decreased. The variations in resistance to thermal denaturation and enzymatic degradation were in accordance with the number of cross-links per unit mass of amniotic membrane, indicating l-lysine-modulated stabilization of collagen molecules. It was also noteworthy that the carbodiimide cross-linked tissue samples prepared using a relatively high l-lysine-pretreated concentration (ie, 30 mM) appeared to have decreased light transmittance and biocompatibility, probably due to the influence of a large nanofiber size and a high charge density. The rise in stemness gene and protein expression levels was dependent on improved cross-link formation, suggesting the crucial role of amino acid bridges in constructing suitable scaffolds to preserve limbal progenitor cells. It is concluded that mild to moderate pretreatment conditions (ie, 3–10 mM l-lysine) can provide a useful strategy to assist in the development of carbodiimide cross-linked amniotic membrane as a stable stem cell niche for corneal epithelial tissue engineering. PMID:25395849

  16. Identification of Potent, Selective, Cell-Active Inhibitors of the Histone Lysine Methyltransferase EZH2.

    PubMed

    Verma, Sharad K; Tian, Xinrong; LaFrance, Louis V; Duquenne, Céline; Suarez, Dominic P; Newlander, Kenneth A; Romeril, Stuart P; Burgess, Joelle L; Grant, Seth W; Brackley, James A; Graves, Alan P; Scherzer, Daryl A; Shu, Art; Thompson, Christine; Ott, Heidi M; Aller, Glenn S Van; Machutta, Carl A; Diaz, Elsie; Jiang, Yong; Johnson, Neil W; Knight, Steven D; Kruger, Ryan G; McCabe, Michael T; Dhanak, Dashyant; Tummino, Peter J; Creasy, Caretha L; Miller, William H

    2012-12-13

    The histone H3-lysine 27 (H3K27) methyltransferase EZH2 plays a critical role in regulating gene expression, and its aberrant activity is linked to the onset and progression of cancer. As part of a drug discovery program targeting EZH2, we have identified highly potent, selective, SAM-competitive, and cell-active EZH2 inhibitors, including GSK926 (3) and GSK343 (6). These compounds are small molecule chemical tools that would be useful to further explore the biology of EZH2. PMID:24900432

  17. Junctional and allele-specific residues are critical for MERS-CoV neutralization by an exceptionally potent germline-like antibody

    DOE PAGESBeta

    Ying, Tianlei; Prabakaran, Ponraj; Du, Lanying; Shi, Wei; Feng, Yang; Wang, Yanping; Wang, Lingshu; Li, Wei; Jiang, Shibo; Dimitrov, Dimiter S.; et al

    2015-09-15

    The MERS-CoV is an emerging virus, which already infected more than 1,300 humans with high (~36%) mortality. Here, we show that m336, an exceptionally potent human anti-MERS-CoV antibody, is almost germline with only one somatic mutation in the heavy chain. The structure of Fab m336 in complex with the MERS-CoV receptor-binding domain reveals that its IGHV1-69-derived heavy chain provides more than 85% binding surface and that its epitope almost completely overlaps with the receptor-binding site. Analysis of antibodies from 69 healthy humans suggests an important role of the V(D)J recombination-generated junctional and allele-specific residues for achieving high affinity of bindingmore » at such low levels of somatic hypermutation. Our results also have important implications for development of vaccine immunogens based on the newly identified m336 epitope as well as for elucidation of mechanisms of neutralization by m336-like antibodies and their elicitation in vivo.« less

  18. Junctional and allele-specific residues are critical for MERS-CoV neutralization by an exceptionally potent germline-like antibody

    SciTech Connect

    Ying, Tianlei; Prabakaran, Ponraj; Du, Lanying; Shi, Wei; Feng, Yang; Wang, Yanping; Wang, Lingshu; Li, Wei; Jiang, Shibo; Dimitrov, Dimiter S.; Zhou, Tongqing

    2015-09-15

    The MERS-CoV is an emerging virus, which already infected more than 1,300 humans with high (~36%) mortality. Here, we show that m336, an exceptionally potent human anti-MERS-CoV antibody, is almost germline with only one somatic mutation in the heavy chain. The structure of Fab m336 in complex with the MERS-CoV receptor-binding domain reveals that its IGHV1-69-derived heavy chain provides more than 85% binding surface and that its epitope almost completely overlaps with the receptor-binding site. Analysis of antibodies from 69 healthy humans suggests an important role of the V(D)J recombination-generated junctional and allele-specific residues for achieving high affinity of binding at such low levels of somatic hypermutation. Our results also have important implications for development of vaccine immunogens based on the newly identified m336 epitope as well as for elucidation of mechanisms of neutralization by m336-like antibodies and their elicitation in vivo.

  19. Small numbers of residual tumor cells at the site of primary inoculation are critical for anti-tumor immunity following challenge at a secondary location.

    PubMed

    Kakinuma, Takashi; Nadiminti, Hari; Lonsdorf, Anke S; Murakami, Takashi; Perez, Bradford A; Kobayashi, Hisataka; Finkelstein, Steven E; Pothiawala, Gulnar; Belkaid, Yasmine; Hwang, Sam T

    2007-07-01

    Luciferase-transduced B16 murine melanoma cells (luc-B16) inoculated in ear skin do not form tumors but prevent tumor formation by luc-B16 cells injected into the footpad. To determine the requirements for such immunity, we followed the fate of luc-B16 cells following ear injection. Surprisingly, small numbers of viable luc-B16 cells were detected in tumor-free mouse skin for up to 60 days post-inoculation. After 1 week, the number of Foxp3(+)CD4(+)CD25(+) T cells (along with foxp3 mRNA expression) increased rapidly in the injected ear skin. Residual tumor cells in ears were reduced in mice treated with anti-CD25 mAb and in CD4-deficient mice, but increased in CD8-deficient mice. Strikingly, the loss of luc-B16 cells in the ear skin, either spontaneously or following amputation of the injected ear, resulted in significantly enhanced tumor formation by parental and luciferase-expressing B16 cells after footpad injection. These studies suggest that small numbers of tumor cells (possibly regulated by CD4(+)CD25(+) regulatory T cells expressing Foxp3) are required for effective host anti-tumor responses at alternate inoculation sites. PMID:17139493

  20. Disulphonic stilbene block of cystic fibrosis transmembrane conductance regulator Cl- channels expressed in a mammalian cell line and its regulation by a critical pore residue.

    PubMed

    Linsdell, P; Hanrahan, J W

    1996-11-01

    1. The disulphonic stilbenes 4,4'-dinitrostilbene-2,2'-disulphonic acid (DNDS) and 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) were shown to cause a voltage-dependent inhibition of macroscopic cystic fibrosis transmembrane conductance regulator (CFTR) Cl- currents expressed in baby hamster kidney cells when applied to the cytoplasmic face of the membrane. These compounds are known to be relatively ineffective at blocking CFTR from the extracellular side of the membrane. 2. Mutation of a positively charged arginine, previously suggested to be located in the channel pore (R347), to a negatively charged aspartate significantly reduced the affinity of block by both DNDS and DIDS, suggesting that this residue contributes to the binding site for disulphonic stilbenes. 3. It is suggested that the CFTR Cl- channel may contain a relatively large inner vestibule in which a number of large anions bind and block Cl- permeation. Arginine 347 may be involved in anion binding within this region. PMID:8930836

  1. Disulphonic stilbene block of cystic fibrosis transmembrane conductance regulator Cl- channels expressed in a mammalian cell line and its regulation by a critical pore residue.

    PubMed Central

    Linsdell, P; Hanrahan, J W

    1996-01-01

    1. The disulphonic stilbenes 4,4'-dinitrostilbene-2,2'-disulphonic acid (DNDS) and 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) were shown to cause a voltage-dependent inhibition of macroscopic cystic fibrosis transmembrane conductance regulator (CFTR) Cl- currents expressed in baby hamster kidney cells when applied to the cytoplasmic face of the membrane. These compounds are known to be relatively ineffective at blocking CFTR from the extracellular side of the membrane. 2. Mutation of a positively charged arginine, previously suggested to be located in the channel pore (R347), to a negatively charged aspartate significantly reduced the affinity of block by both DNDS and DIDS, suggesting that this residue contributes to the binding site for disulphonic stilbenes. 3. It is suggested that the CFTR Cl- channel may contain a relatively large inner vestibule in which a number of large anions bind and block Cl- permeation. Arginine 347 may be involved in anion binding within this region. PMID:8930836

  2. Targeting of pro-apoptotic TLR adaptor SARM to mitochondria: definition of the critical region and residues in the signal sequence.

    PubMed

    Panneerselvam, Porkodi; Singh, Laishram Pradeepkumar; Ho, Bow; Chen, Jianzhu; Ding, Jeak Ling

    2012-03-01

    The fifth and the most well-conserved member of the TLR (Toll-like receptor) adaptor, SARM (sterile α- and HEAT/armadillo-motif-containing protein), has been reported to be an important mediator of apoptosis. However, the exact cellular localization of SARM with respect to its role is unclear. In the present study we show that SARM specifically co-localizes with mitochondria. Endogenous SARM is mainly found in the mitochondria. We demonstrate that the N-terminal 27 amino acids (S27) of SARM, which is hydrophobic and polybasic, acts as a mitochondria-targeting signal sequence, associating SARM to the mitochondria. The S27 peptide has an inherent ability to bind to lipids and mitochondria. This sequence effectively translocates the soluble EGFP (enhanced green fluorescence protein) reporter into the mitochondria. Positioning S27 downstream of the EGFP abrogates its mitochondria-targeting ability. Transmission electron microscopy confirms the ability of S27 to import EGFP into the mitochondria. Importantly, by mutagenesis study, we delineated the specificity of the mitochondria-targeting ability to the arginine residue at the 14th position. The R14A SARM mutant also showed reduced apoptotic potential when compared with the wild-type. Taken together, S27, which is a bona fide signal sequence that targets SARM to the mitochondria, explains the pro-apoptotic activity of SARM. PMID:22145856

  3. Junctional and allele-specific residues are critical for MERS-CoV neutralization by an exceptionally potent germline-like antibody

    PubMed Central

    Ying, Tianlei; Prabakaran, Ponraj; Du, Lanying; Shi, Wei; Feng, Yang; Wang, Yanping; Wang, Lingshu; Li, Wei; Jiang, Shibo; Dimitrov, Dimiter S.; Zhou, Tongqing

    2015-01-01

    The MERS-CoV is an emerging virus, which already infected more than 1,300 humans with high (∼36%) mortality. Here, we show that m336, an exceptionally potent human anti-MERS-CoV antibody, is almost germline with only one somatic mutation in the heavy chain. The structure of Fab m336 in complex with the MERS-CoV receptor-binding domain reveals that its IGHV1-69-derived heavy chain provides more than 85% binding surface and that its epitope almost completely overlaps with the receptor-binding site. Analysis of antibodies from 69 healthy humans suggests an important role of the V(D)J recombination-generated junctional and allele-specific residues for achieving high affinity of binding at such low levels of somatic hypermutation. Our results also have important implications for development of vaccine immunogens based on the newly identified m336 epitope as well as for elucidation of mechanisms of neutralization by m336-like antibodies and their elicitation in vivo. PMID:26370782

  4. HISTONE LYSINE DEMETHYLASES IN BREAST CANCER

    PubMed Central

    Paolicchi, Elisa; Crea, Francesco; Farrar, William L; Green, Jeffrey E; Danesi, Romano

    2013-01-01

    Histone lysine demethylases (KDMs) have been recently discovered in mammals and have been nicknamed “erasers” for their ability to remove methyl groups from histone substrates. In cancer cells, KDMs can activate or repress gene transcription, behaving as oncogenes or tumor suppressors depending upon the cellular context. In order to investigate the potential role of KDMs in Breast Cancer (BC), we queried the Oncomine database and determined that the expression of KDMs correlates with BC prognosis. High expression of KDM3B and KDM5A is associated with a better prognosis (no recurrence after mastectomy p=0.005 and response to docetaxel p=0.005); conversely, KDM6A is overexpressed in BC patients with an unfavorable prognosis (mortality at 1 year, p=8.65E-7). Our findings suggest that KDMs could be potential targets for BC therapy. Further, altering the interactions between KDMs and Polycomb Group genes (PcG) may provide novel avenues for therapy that specifically targets these genes in BC. PMID:23266085

  5. N-epsilon-(carboxyethyl)lysine, a product of the chemical modification of proteins by methylglyoxal, increases with age in human lens proteins.

    PubMed Central

    Ahmed, M U; Brinkmann Frye, E; Degenhardt, T P; Thorpe, S R; Baynes, J W

    1997-01-01

    Advanced glycation end-products and glycoxidation products, such as Nepsilon-(carboxymethyl)lysine (CML) and pentosidine, accumulate in long-lived tissue proteins with age and are implicated in the aging of tissue proteins and in the development of pathology in diabetes, atherosclerosis and other diseases. In this paper we describe a new advanced glycation end-product, Nepsilon-(carboxyethyl)lysine (CEL), which is formed during the reaction of methylglyoxal with lysine residues in model compounds and in the proteins RNase and collagen. CEL was also detected in human lens proteins at a concentration similar to that of CML, and increased with age in parallel with the concentration of CML. Although CEL was formed in highest yields during the reaction of methylglyoxal and triose phosphates with lysine and protein, it was also formed in reactions of pentoses, ascorbate and other sugars with lysine and RNase. We propose that levels of CML and CEL and their ratio to one another in tissue proteins and in urine will provide an index of glyoxal and methylglyoxal concentrations in tissues, alterations in glutathione homoeostasis and dicarbonyl metabolism in disease, and sources of advanced glycation end-products in tissue proteins in aging and disease. PMID:9182719

  6. Water reuse in the l-lysine fermentation process

    SciTech Connect

    Hsiao, T.Y.; Glatz, C.E.

    1996-02-05

    L-Lysine is produced commercially by fermentation. As is typical for fermentation processes, a large amount of liquid waste is generated. To minimize the waste, which is mostly the broth effluent from the cation exchange column used for l-lysine recovery, the authors investigated a strategy of recycling a large fraction of this broth effluent to the subsequent fermentation. This was done on a lab-scale process with Corynebacterium glutamicum ATCC 21253 as the l-lysine-producing organisms. Broth effluent from a fermentation in a defined medium was able to replace 75% of the water for the subsequent batch; this recycle ratio was maintained for 3 sequential batches without affecting cell mass and l-lysine production. Broth effluent was recycled at 50% recycle ratio in a fermentation in a complex medium containing beet molasses. The first recycle batch had an 8% lower final l-lysine level, but 8% higher maximum cell mass. In addition to reducing the volume of liquid waste, this recycle strategy has the additional advantage of utilizing the ammonium desorbed from the ion-exchange column as a nitrogen source in the recycle fermentation. The major problem of recycling the effluent from the complex medium was in the cation-exchange operation, where column capacity was 17% lower for the recycle batch. The loss of column capacity probably results from the buildup of cations competing with l-lysine for binding.

  7. Biofortification of rice with lysine using endogenous histones.

    PubMed

    Wong, H W; Liu, Q; Sun, S S M

    2015-02-01

    Rice is the most consumed cereal grain in the world, but deficient in the essential amino acid lysine. Therefore, people in developing countries with limited food diversity who rely on rice as their major food source may suffer from malnutrition. Biofortification of stable crops by genetic engineering provides a fast and sustainable method to solve this problem. In this study, two endogenous rice lysine-rich histone proteins, RLRH1 and RLRH2, were over-expressed in rice seeds to achieve lysine biofortification. Their protein sequences passed an allergic sequence-based homology test. Their accumulations in rice seeds were raised to a moderate level by the use of a modified rice glutelin 1 promoter with lowered expression strength to avoid the occurrence of physiological abnormalities like unfolded protein response. The expressed proteins were further targeted to protein storage vacuoles for stable storage using a glutelin 1 signal peptide. The lysine content in the transgenic rice seeds was enhanced by up to 35 %, while other essential amino acids remained balanced, meeting the nutritional standards of the World Health Organization. No obvious unfolded protein response was detected. Different degrees of chalkiness, however, were detected in the transgenic seeds, and were positively correlated with both the levels of accumulated protein and lysine enhancement. This study offered a solution to the lysine deficiency in rice, while at the same time addressing concerns about food safety and physiological abnormalities in biofortified crops. PMID:25512028

  8. Insights into the regulatory landscape of the lysine riboswitch

    PubMed Central

    Garst, Andrew D.; Porter, Ely B.; Batey, Robert T.

    2012-01-01

    A prevalent means of regulating gene expression in bacteria is by riboswitches found within mRNA leader sequences. Like protein repressors these RNA elements must bind an effector molecule with high specificity against a background of other cellular metabolites of similar chemical structure to elicit the appropriate regulatory response. Current crystal structures of the lysine riboswitch do not provide a complete understanding of selectivity as recognition is substantially mediated through main chain atoms of the amino acid. Using a directed set of lysine analogs and other amino acids, the relative contributions of the polar functional groups to binding affinity and the regulatory response have been determined. Our results reveal that the lysine riboswitch has >1,000-fold specificity for lysine over other amino acids. To achieve this specificity, the aptamer is highly sensitive to the precise placement of the ε-amino group and relatively tolerant of alterations to the main chain functional groups. At low NTP concentrations, we observe good agreement between the half-maximal regulatory activity (T50) and the affinity of the receptor for lysine (KD) as well many of its analogs. However, above 400 µM [NTP] the concentration of lysine required to elicit transcription termination rises, moving into the riboswitch into a kinetic control regime. These data demonstrate that under physiologically relevant conditions riboswitches can integrate both effector and NTP concentrations to generate a regulatory response appropriate for global metabolic state of the cell. PMID:22771573

  9. Insights into the regulatory landscape of the lysine riboswitch.

    PubMed

    Garst, Andrew D; Porter, Ely B; Batey, Robert T

    2012-10-12

    A prevalent means of regulating gene expression in bacteria is by riboswitches found within mRNA leader sequences. Like protein repressors, these RNA elements must bind an effector molecule with high specificity against a background of other cellular metabolites of similar chemical structure to elicit the appropriate regulatory response. Current crystal structures of the lysine riboswitch do not provide a complete understanding of selectivity as recognition is substantially mediated through main-chain atoms of the amino acid. Using a directed set of lysine analogs and other amino acids, we have determined the relative contributions of the polar functional groups to binding affinity and the regulatory response. Our results reveal that the lysine riboswitch has >1000-fold specificity for lysine over other amino acids. The aptamer is highly sensitive to the precise placement of the ε-amino group and relatively tolerant of alterations to the main-chain functional groups in order to achieve this specificity. At low nucleotide triphosphate (NTP) concentrations, we observe good agreement between the half-maximal regulatory activity (T(50)) and the affinity of the receptor for lysine (K(d)), as well as many of its analogs. However, above 400 μM [NTP], the concentration of lysine required to elicit transcription termination rises, moving into the riboswitch into a kinetic control regime. These data demonstrate that, under physiologically relevant conditions, riboswitches can integrate both effector and NTP concentrations to generate a regulatory response appropriate for global metabolic state of the cell. PMID:22771573

  10. The Draft Genome and Transcriptome of Amaranthus hypochondriacus: A C4 Dicot Producing High-Lysine Edible Pseudo-Cereal

    PubMed Central

    Sunil, Meeta; Hariharan, Arun K.; Nayak, Soumya; Gupta, Saurabh; Nambisan, Suran R.; Gupta, Ravi P.; Panda, Binay; Choudhary, Bibha; Srinivasan, Subhashini

    2014-01-01

    Grain amaranths, edible C4 dicots, produce pseudo-cereals high in lysine. Lysine being one of the most limiting essential amino acids in cereals and C4 photosynthesis being one of the most sought-after phenotypes in protein-rich legume crops, the genome of one of the grain amaranths is likely to play a critical role in crop research. We have sequenced the genome and transcriptome of Amaranthus hypochondriacus, a diploid (2n = 32) belonging to the order Caryophyllales with an estimated genome size of 466 Mb. Of the 411 linkage single-nucleotide polymorphisms (SNPs) reported for grain amaranths, 355 SNPs (86%) are represented in the scaffolds and 74% of the 8.6 billion bases of the sequenced transcriptome map to the genomic scaffolds. The genome of A. hypochondriacus, codes for at least 24,829 proteins, shares the paleohexaploidy event with species under the superorders Rosids and Asterids, harbours 1 SNP in 1,000 bases, and contains 13.76% of repeat elements. Annotation of all the genes in the lysine biosynthetic pathway using comparative genomics and expression analysis offers insights into the high-lysine phenotype. As the first grain species under Caryophyllales and the first C4 dicot genome reported, the work presented here will be beneficial in improving crops and in expanding our understanding of angiosperm evolution. PMID:25071079

  11. The Frequency of 1,4-Benzoquinone-Lysine Adducts in Cytochrome c Correlate with Defects in Apoptosome Activation

    PubMed Central

    Fisher, Ashley A.; Labenski, Matthew T.; Chapman, John D.; Bratton, Shawn B.; Monks, Terrence J.; Lau, Serrine S.

    2011-01-01

    Electrophile-mediated post-translational modifications (PTMs) are known to cause tissue toxicities and disease progression. These effects are mediated via site-specific modifications and structural disruptions associated with such modifications. 1,4-Benzoquinone (BQ) and its quinone-thioether metabolites are electrophiles that elicit their toxicity via protein arylation and the generation of reactive oxygen species. Site-specific BQ-lysine adducts are found on residues in cytochrome c that are necessary for protein-protein interactions, and these adducts contribute to interferences in its ability to facilitate apoptosome formation. To further characterize the structural and functional impact of these BQ-mediated PTMs, the original mixture of BQ-adducted cytochrome c was fractionated by liquid isoelectric focusing to provide various fractions of BQ-adducted cytochrome c species devoid of the native protein. The fractionation process separates samples based on their isoelectric point (pI), and because BQ adducts form predominantly on lysine residues, increased numbers of BQ adducts on cytochrome c correlate with a lower protein pI. Each fraction was analyzed for structural changes, and each was also assayed for the ability to support apoptosome-mediated activation of caspase-3. Circular dichroism revealed that several of the BQ-adducted cytochrome c species maintained a slightly more rigid structure in comparison to native cytochrome c. BQ-adducted cytochrome c also failed to activate caspase-3, with increasing numbers of BQ-lysine adducts corresponding to a greater inability to activate the apoptosome. In summary, the specific site of the BQ-lysine adducts, and the nature of the adduct, are important determinants of the subsequent structural changes to cytochrome c. In particular, adducts at sites necessary for protein-protein interactions interfere with the proapoptotic function of cytochrome c. PMID:21527774

  12. N-Ethyl-N-Nitrosourea (ENU) Mutagenesis Reveals an Intronic Residue Critical for Caenorhabditis elegans 3' Splice Site Function in Vivo.

    PubMed

    Itani, Omar A; Flibotte, Stephane; Dumas, Kathleen J; Guo, Chunfang; Blumenthal, Thomas; Hu, Patrick J

    2016-01-01

    Metazoan introns contain a polypyrimidine tract immediately upstream of the AG dinucleotide that defines the 3' splice site. In the nematode Caenorhabditis elegans, 3' splice sites are characterized by a highly conserved UUUUCAG/R octamer motif. While the conservation of pyrimidines in this motif is strongly suggestive of their importance in pre-mRNA splicing, in vivo evidence in support of this is lacking. In an N-ethyl-N-nitrosourea (ENU) mutagenesis screen in Caenorhabditis elegans, we have isolated a strain containing a point mutation in the octamer motif of a 3' splice site in the daf-12 gene. This mutation, a single base T-to-G transversion at the -5 position relative to the splice site, causes a strong daf-12 loss-of-function phenotype by abrogating splicing. The resulting transcript is predicted to encode a truncated DAF-12 protein generated by translation into the retained intron, which contains an in-frame stop codon. Other than the perfectly conserved AG dinucleotide at the site of splicing, G at the -5 position of the octamer motif is the most uncommon base in C. elegans 3' splice sites, occurring at closely paired sites where the better match to the splicing consensus is a few bases downstream. Our results highlight both the biological importance of the highly conserved -5 uridine residue in the C. elegans 3' splice site octamer motif as well as the utility of using ENU as a mutagen to study the function of polypyrimidine tracts and other AU- or AT-rich motifs in vivo. PMID:27172199

  13. N-Ethyl-N-Nitrosourea (ENU) Mutagenesis Reveals an Intronic Residue Critical for Caenorhabditis elegans 3′ Splice Site Function in Vivo

    PubMed Central

    Itani, Omar A.; Flibotte, Stephane; Dumas, Kathleen J.; Guo, Chunfang; Blumenthal, Thomas; Hu, Patrick J.

    2016-01-01

    Metazoan introns contain a polypyrimidine tract immediately upstream of the AG dinucleotide that defines the 3′ splice site. In the nematode Caenorhabditis elegans, 3′ splice sites are characterized by a highly conserved UUUUCAG/R octamer motif. While the conservation of pyrimidines in this motif is strongly suggestive of their importance in pre-mRNA splicing, in vivo evidence in support of this is lacking. In an N-ethyl-N-nitrosourea (ENU) mutagenesis screen in Caenorhabditis elegans, we have isolated a strain containing a point mutation in the octamer motif of a 3′ splice site in the daf-12 gene. This mutation, a single base T-to-G transversion at the -5 position relative to the splice site, causes a strong daf-12 loss-of-function phenotype by abrogating splicing. The resulting transcript is predicted to encode a truncated DAF-12 protein generated by translation into the retained intron, which contains an in-frame stop codon. Other than the perfectly conserved AG dinucleotide at the site of splicing, G at the –5 position of the octamer motif is the most uncommon base in C. elegans 3′ splice sites, occurring at closely paired sites where the better match to the splicing consensus is a few bases downstream. Our results highlight both the biological importance of the highly conserved –5 uridine residue in the C. elegans 3′ splice site octamer motif as well as the utility of using ENU as a mutagen to study the function of polypyrimidine tracts and other AU- or AT-rich motifs in vivo. PMID:27172199

  14. A mutagenic study identifying critical residues for the structure and function of rice manganese transporter OsMTP8.1

    PubMed Central

    Chen, Xi; Li, Jiyu; Wang, Lihua; Ma, Gang; Zhang, Wei

    2016-01-01

    Rice (Oryza sativa) MTP8.1 (OsMTP8.1) is a tonoplast-localized manganese transporter of the cation diffusion facilitator family. Here we present a structure-function analysis of OsMTP8.1 based on the site-directed and random mutagenesis and complementation assays in manganese hypersensitive yeast, in combination with three-dimensional (3D) structure modeling based on the crystal structure of the Escherichia coli CDF family member, EcYiiP. Two metal-binding sites are conserved in OsMTP8.1 with EcYiiP, one is between transmembrane helices TM2 and TM5, the other is the cytoplasmic C-terminus. In addition to these two metal-binding sites, there may exist other Mn-binding sites such as that at the very end of the CTD. Two residues (R167 and L296) may play an important role for the hinge-like movement of CTDs. Several mutations such as E357A and V374D may affect dimer formation, and S132A may induce a conformational change, resulting in a loss of transport function or modification in metal selectivity. The N-terminus of OsMTP8.1 was not functional for Mn transport activity, and the real function of NTD remains to be investigated in the future. The findings of the present study illustrate the structure-function relationship of OsMTP8.1 in Mn transport activity, which may also be applied to other plant Mn-CDF proteins. PMID:27555514

  15. Topological dispositions of lysine. alpha. 380 and lysine. gamma. 486 in the acetylcholine receptor from Torpedo californica

    SciTech Connect

    Dwyer, B.P. )

    1991-04-23

    The locations have been determined, with respect to the plasma membrane, of lysine {alpha}380 and lysine {gamma}486 in the {alpha} subunit and the {gamma} subunit, respectively, of the nicotinic acetylcholine receptor from Torpedo californica. Immunoadsorbents were constructed that recognize the carboxy terminus of the peptide GVKYIAE released by proteolytic digestion from positions 378-384 in the amino acid sequence of the {alpha} subunit of the acetylcholine receptor and the carboxy terminus of the peptide KYVP released by proteolytic digestion from positions 486-489 in the amino acid sequence of the {gamma} subunit. They were used to isolate these peptides from proteolytic digests of polypeptides from the acetylcholine receptor. Sealed vesicles containing the native acetylcholine receptor were labeled with pyridoxal phosphate and sodium ({sup 3}H)-borohydride. The effect of saponin on the incorporation of pyridoxamine phosphate into lysine {alpha}380 and lysine {gamma}486 from the acetylcholine receptor in these vesicles was assessed with the immunoadsorbents. The conclusions that follow from these results are that lysine {alpha}380 is on the inside surface of a vesicle and lysine {gamma}486 is on the outside surface. Because a majority (85%) of the total binding sites for {alpha}-bungarotoxin bind the toxin in the absence of saponin, the majority of the vesicles are right side out with the inside of the vesicle corresponding to the cytoplasmic surface and the outside of the vesicle corresponding to the extracytoplasmic, synaptic surface. Because lysine {alpha}380 and lysine {gamma}486 lie on opposite sides of the membrane, a membrane-spanning segment must be located between the two positions occupied by these two amino acids in the common sequence of a polypeptide of the acetylcholine receptor.

  16. Mitochondrial localization of fission yeast manganese superoxide dismutase is required for its lysine acetylation and for cellular stress resistance and respiratory growth

    SciTech Connect

    Takahashi, Hidekazu; Shirai, Atsuko; Matsuyama, Akihisa; Yoshida, Minoru

    2011-03-04

    Research highlights: {yields} Fission yeast manganese superoxide dismutase (MnSOD) is acetylated. {yields} The mitochondrial targeting sequence (MTS) is required for the acetylation of MnSOD. {yields} The MTS is not crucial for MnSOD activity, but is important for respiratory growth. {yields} Posttranslational regulation of MnSOD differs between budding and fission yeast. -- Abstract: Manganese-dependent superoxide dismutase (MnSOD) is localized in the mitochondria and is important for oxidative stress resistance. Although transcriptional regulation of MnSOD has been relatively well studied, much less is known about the protein's posttranslational regulation. In budding yeast, MnSOD is activated after mitochondrial import by manganese ion incorporation. Here we characterize posttranslational modification of MnSOD in the fission yeast Schizosaccharomyces pombe. Fission yeast MnSOD is acetylated at the 25th lysine residue. This acetylation was diminished by deletion of N-terminal mitochondrial targeting sequence, suggesting that MnSOD is acetylated after import into mitochondria. Mitochondrial localization of MnSOD is not essential for the enzyme activity, but is crucial for oxidative stress resistance and growth under respiratory conditions of fission yeast. These results suggest that, unlike the situation in budding yeast, S. pombe MnSOD is already active even before mitochondrial localization; nonetheless, mitochondrial localization is critical to allow the cell to cope with reactive oxygen species generated inside or outside of mitochondria.

  17. Use of a tritium release assay to measure 6-N-trimethyl-L-lysine hydroxylase activity: synthesis of 6-N-(3-/sup 3/H)Trimethyl-DL-lysine

    SciTech Connect

    Stein, R.; England, S.

    1981-09-01

    6-N-(3-/sup 3/H)Trimethyl-DL-lysine was synthesized from 6-N-acetyl-L-lysine by the following chemical scheme: 6-N-acetyl-L-lysine ..-->.. 2-keto-6-N-acetylcaproic acid ..-->.. 2-(3-/sup 3/H)keto-6-N-acetylcaproic acid ..-->.. 2-(3-/sup 3/H)keto-6-N-acetylcaproic acid oxime ..-->.. 6-N-(3-/sup 3/H)acetyl-DL-lysine ..-->.. DL-(3-/sup 3/H)lysine ..-->.. 2-N-(3-/sup 3/H)formyl-DL-lysine ..-->.. 2-(3-/sup 3/H)formyl-6-N-trimethyl-DL-lysine ..-->.. 6-N-(3-/sup 3/H)trimethyl-DL-lysine. Using a 70% ammonium sulfate fraction obtained from a high-speed rate kidney supernatant, the cosubstrate and cofactor requirements for 6-N-trimethyl-L-lysine hydroxylase activity as measured by tritium release from 6-N-(3-/sup 3/H)trimethyl-DL-lysine were: ..cap alpha..-ketoglutarate, ferrous ions, L-ascorbate, and oxygen, with added catalase showing a slight but distinct stimulatory effect. On incubation with the crude rat kidney preparation, the release of tritium from 6-N-(3-/sup 3/H)trimethyl-DL-lysine was linear with both time of incubation and protein concentration. Hydroxylation of 6-N-trimethyl-L-lysine, as measured by tritium release from the labeled substrate, was examined in rat kidney, heart, liver, and skeletal muscle tissues, and found to be most active in the kidney.

  18. Metabolic fluxes in Corynebacterium glutamicum during lysine production with sucrose as carbon source.

    PubMed

    Wittmann, Christoph; Kiefer, Patrick; Zelder, Oskar

    2004-12-01

    Metabolic fluxes in the central metabolism were determined for lysine-producing Corynebacterium glutamicum ATCC 21526 with sucrose as a carbon source, providing an insight into molasses-based industrial production processes with this organism. For this purpose, 13C metabolic flux analysis with parallel studies on [1-(13C)Fru]sucrose, [1-(13C)Glc]sucrose, and [13C6Fru]sucrose was carried out. C. glutamicum directed 27.4% of sucrose toward extracellular lysine. The strain exhibited a relatively high flux of 55.7% (normalized to an uptake flux of hexose units of 100%) through the pentose phosphate pathway (PPP). The glucose monomer of sucrose was completely channeled into the PPP. After transient efflux, the fructose residue was mainly taken up by the fructose-specific phosphotransferase system (PTS) and entered glycolysis at the level of fructose-1,6-bisphosphate. Glucose-6-phosphate isomerase operated in the gluconeogenetic direction from fructose-6-phosphate to glucose-6-phosphate and supplied additional carbon (7.2%) from the fructose part of the substrate toward the PPP. This involved supply of fructose-6-phosphate from the fructose part of sucrose either by PTS(Man) or by fructose-1,6-bisphosphatase. C. glutamicum further exhibited a high tricarboxylic acid (TCA) cycle flux of 78.2%. Isocitrate dehydrogenase therefore significantly contributed to the total NADPH supply of 190%. The demands for lysine (110%) and anabolism (32%) were lower than the supply, resulting in an apparent NADPH excess. The high TCA cycle flux and the significant secretion of dihydroxyacetone and glycerol display interesting targets to be approached by genetic engineers for optimization of the strain investigated. PMID:15574927

  19. [Cloning, prokaryotic expression and characterization of lysine decarboxylase gene from Huperzia serrata].

    PubMed

    Di, Ci; Li, Jing; Tang, Yuntao; Peng, Qingzhong

    2014-08-01

    Huperzine A is a promising drug to treat Alzheimer's disease (AD). To date, its biosynthetic pathway is still unknown. Lysine decarboxylase (LDC) has been proposed to catalyze the first-step of the biosynthesis of huperzine A. To identify and characterize LDCs from Huperzia serrata, we isolated two LDC fragments (LDC1 and LDC2) from leaves of H. serrata by RT-PCR and then cloned them into pMD 19-T vector. Sequence analysis showed that LDC1 and LDC2 genes shared 95.3% identity and encoded the protein of 212 and 202 amino acid residues respectively. Thus, we ligated LDC genes into pET-32a(+) to obtain recombinant expressing vectors pET-32a(+)/LDC1 and pET-32a(+)/LDC2 respectively. We further introduced two expression vectors into Escherichia coli BL21(DE3) and cultured positive colonies of E. coli in liquid LB medium. After inducing for 4 hours with 260 μg/mL IPTG at 30 degrees C, soluble recombinant Trx-LDC1 and Trx-LDC2 were obtained and isolated for purification using a Ni-NTA affinity chromatography. We incubated purified recombinant proteins with L-lysine in the enzyme reaction buffer at 37 degrees C and then derived the reaction products using dansyl chloride. It was found that both Trx-LDC1 and Trx-LDC2 had decarboxylase activity, could convert L-lysine into cadaverine by way of thin layer chromatography assay. Further, bioinformatics analysis indicated that deduced LDC1 and LDC2 had different physicochemical properties, but similar secondary and three-dimensional structures. PMID:25423760

  20. [Cloning, prokaryotic expression and characterization of lysine decarboxylase gene from Huperzia serrata].

    PubMed

    Di, Ci; Li, Jing; Tang, Yuntao; Peng, Qingzhong

    2014-08-01

    Huperzine A is a promising drug to treat Alzheimer's disease (AD). To date, its biosynthetic pathway is still unknown. Lysine decarboxylase (LDC) has been proposed to catalyze the first-step of the biosynthesis of huperzine A. To identify and characterize LDCs from Huperzia serrata, we isolated two LDC fragments (LDC1 and LDC2) from leaves of H. serrata by RT-PCR and then cloned them into pMD 19-T vector. Sequence analysis showed that LDC1 and LDC2 genes shared 95.3% identity and encoded the protein of 212 and 202 amino acid residues respectively. Thus, we ligated LDC genes into pET-32a(+) to obtain recombinant expressing vectors pET-32a(+)/LDC1 and pET-32a(+)/LDC2 respectively. We further introduced two expression vectors into Escherichia coli BL21(DE3) and cultured positive colonies of E. coli in liquid LB medium. After inducing for 4 hours with 260 μg/mL IPTG at 30 degrees C, soluble recombinant Trx-LDC1 and Trx-LDC2 were obtained and isolated for purification using a Ni-NTA affinity chromatography. We incubated purified recombinant proteins with L-lysine in the enzyme reaction buffer at 37 degrees C and then derived the reaction products using dansyl chloride. It was found that both Trx-LDC1 and Trx-LDC2 had decarboxylase activity, could convert L-lysine into cadaverine by way of thin layer chromatography assay. Further, bioinformatics analysis indicated that deduced LDC1 and LDC2 had different physicochemical properties, but similar secondary and three-dimensional structures. PMID:25507483

  1. Cancers and the NSD family of histone lysine methyltransferases.

    PubMed

    Morishita, Masayo; di Luccio, Eric

    2011-12-01

    Both genetic and epigenetic alterations are responsible for the stepwise initiation and progression of cancers. Only epigenetic aberrations can be reversible, allowing the malignant cell population to revert to a more benign phenotype. The epigenetic therapy of cancers is emerging as an effective and valuable approach to both the chemotherapy and the chemoprevention of cancer. The utilization of epigenetic targets that include histone methyltransferase (HMTase), Histone deacetylatase, and DNA methyltransferase, are emerging as key therapeutic targets. The nuclear receptor binding SET domain (NSD) protein is a family of three HMTases, NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L1, and plays a critical part in chromatin integrity as evidenced by a growing number of conditions linked to the alterations and/or amplification of NSD1, NSD2, and/or NSD3. NSD1, NSD2 and NSD3 are associated with multiple cancers. The amplification of either NSD1 or NSD2 triggers the cellular transformation and thus is key in the early carcinogenesis events. In most cases, reducing the levels of NSD proteins would suppress cancer growth. NSD1 and NSD2 were isolated as genes linked to developmental diseases, such as Sotos syndrome and Wolf-Hirschhorn syndrome, respectively, implying versatile aspects of the NSD proteins. The NSD pathways, however, are not well understood. It is noteworthy that the NSD family is phylogenetically distinct compared to other known lysine-HMTases, Here, we review the current knowledge on NSD1/NSD2/NSD3 in tumorigenesis and prospect their special value for developing novel anticancer drugs. PMID:21664949

  2. Crystal Structure of the Lysine Riboswitch Regulatory mRNA Element*S⃞

    PubMed Central

    Garst, Andrew D.; Héroux, Annie; Rambo, Robert P.; Batey, Robert T.

    2008-01-01

    Riboswitches are metabolite-sensitive elements found in mRNAs that control gene expression through a regulatory secondary structural switch. Along with regulation of lysine biosynthetic genes, mutations within the lysine-responsive riboswitch (L-box) play a role in the acquisition of resistance to antimicrobial lysine analogs. To understand the structural basis for lysine binding, we have determined the 2.8Å resolution crystal structure of lysine bound to the Thermotoga maritima asd lysine riboswitch ligand-binding domain. The structure reveals a complex architecture scaffolding a binding pocket completely enveloping lysine. Mutations conferring antimicrobial resistance cluster around this site as well as highly conserved long range interactions, indicating that they disrupt lysine binding or proper folding of the RNA. Comparison of the free and bound forms by x-ray crystallography, small angle x-ray scattering, and chemical probing reveals almost identical structures, indicating that lysine induces only limited and local conformational changes upon binding. PMID:18593706

  3. Crystal structure of the lysine riboswitch regulatory mRNA element.

    PubMed

    Garst, Andrew D; Héroux, Annie; Rambo, Robert P; Batey, Robert T

    2008-08-15

    Riboswitches are metabolite-sensitive elements found in mRNAs that control gene expression through a regulatory secondary structural switch. Along with regulation of lysine biosynthetic genes, mutations within the lysine-responsive riboswitch (L-box) play a role in the acquisition of resistance to antimicrobial lysine analogs. To understand the structural basis for lysine binding, we have determined the 2.8 angstroms resolution crystal structure of lysine bound to the Thermotoga maritima asd lysine riboswitch ligand-binding domain. The structure reveals a complex architecture scaffolding a binding pocket completely enveloping lysine. Mutations conferring antimicrobial resistance cluster around this site as well as highly conserved long range interactions, indicating that they disrupt lysine binding or proper folding of the RNA. Comparison of the free and bound forms by x-ray crystallography, small angle x-ray scattering, and chemical probing reveals almost identical structures, indicating that lysine induces only limited and local conformational changes upon binding. PMID:18593706

  4. Crystal Structure of the Lysine Riboswitch Regulatory mRNA Element

    SciTech Connect

    Garst, A.; Heroux, A; Rambo, R; Batey, R

    2008-01-01

    Riboswitches are metabolite-sensitive elements found in mRNAs that control gene expression through a regulatory secondary structural switch. Along with regulation of lysine biosynthetic genes, mutations within the lysine-responsive riboswitch (L-box) play a role in the acquisition of resistance to antimicrobial lysine analogs. To understand the structural basis for lysine binding, we have determined the 2.8{angstrom} resolution crystal structure of lysine bound to the Thermotoga maritima asd lysine riboswitch ligand-binding domain. The structure reveals a complex architecture scaffolding a binding pocket completely enveloping lysine. Mutations conferring antimicrobial resistance cluster around this site as well as highly conserved long range interactions, indicating that they disrupt lysine binding or proper folding of the RNA. Comparison of the free and bound forms by x-ray crystallography, small angle x-ray scattering, and chemical probing reveals almost identical structures, indicating that lysine induces only limited and local conformational changes upon binding.

  5. Coevolutionary analysis enabled rational deregulation of allosteric enzyme inhibition in Corynebacterium glutamicum for lysine production.

    PubMed

    Chen, Zhen; Meyer, Weiqian; Rappert, Sugima; Sun, Jibin; Zeng, An-Ping

    2011-07-01

    Product feedback inhibition of allosteric enzymes is an essential issue for the development of highly efficient microbial strains for bioproduction. Here we used aspartokinase from Corynebacterium glutamicum (CgAK), a key enzyme controlling the biosynthesis of industrially important aspartate family amino acids, as a model to demonstrate a fast and efficient approach to the deregulation of allostery. In the last 50 years many researchers and companies have made considerable efforts to deregulate this enzyme from allosteric inhibition by lysine and threonine. However, only a limited number of positive mutants have been identified so far, almost exclusively by random mutation and selection. In this study, we used statistical coupling analysis of protein sequences, a method based on coevolutionary analysis, to systematically clarify the interaction network within the regulatory domain of CgAK that is essential for allosteric inhibition. A cluster of interconnected residues linking different inhibitors' binding sites as well as other regions of the protein have been identified, including most of the previously reported positions of successful mutations. Beyond these mutation positions, we have created another 14 mutants that can partially or completely desensitize CgAK from allosteric inhibition, as shown by enzyme activity assays. The introduction of only one of the inhibition-insensitive CgAK mutations (here Q298G) into a wild-type C. glutamicum strain by homologous recombination resulted in an accumulation of 58 g/liter L-lysine within 30 h of fed-batch fermentation in a bioreactor. PMID:21531824

  6. Acetylation mimic of lysine 280 exacerbates human Tau neurotoxicity in vivo

    PubMed Central

    Gorsky, Marianna Karina; Burnouf, Sylvie; Dols, Jacqueline; Mandelkow, Eckhard; Partridge, Linda

    2016-01-01

    Dysfunction and accumulation of the microtubule-associated human Tau (hTau) protein into intraneuronal aggregates is observed in many neurodegenerative disorders including Alzheimer’s disease (AD). Reversible lysine acetylation has recently emerged as a post-translational modification that may play an important role in the modulation of hTau pathology. Acetylated hTau species have been observed within hTau aggregates in human AD brains and multi-acetylation of hTau in vitro regulates its propensity to aggregate. However, whether lysine acetylation at position 280 (K280) modulates hTau-induced toxicity in vivo is unknown. We generated new Drosophila transgenic models of hTau pathology to evaluate the contribution of K280 acetylation to hTau toxicity, by analysing the respective toxicity of pseudo-acetylated (K280Q) and pseudo-de-acetylated (K280R) mutant forms of hTau. We observed that mis-expression of pseudo-acetylated K280Q-hTau in the adult fly nervous system potently exacerbated fly locomotion defects and photoreceptor neurodegeneration. In addition, modulation of K280 influenced total hTau levels and phosphorylation without changing hTau solubility. Altogether, our results indicate that pseudo-acetylation of the single K280 residue is sufficient to exacerbate hTau neurotoxicity in vivo, suggesting that acetylated K280-hTau species contribute to the pathological events leading to neurodegeneration in AD. PMID:26940749

  7. The Helicase Activity of Hyperthermophilic Archaeal MCM is Enhanced at High Temperatures by Lysine Methylation

    PubMed Central

    Xia, Yisui; Niu, Yanling; Cui, Jiamin; Fu, Yang; Chen, Xiaojiang S.; Lou, Huiqiang; Cao, Qinhong

    2015-01-01

    Lysine methylation and methyltransferases are widespread in the third domain of life, archaea. Nevertheless, the effects of methylation on archaeal proteins wait to be defined. Here, we report that recombinant sisMCM, an archaeal homolog of Mcm2-7 eukaryotic replicative helicase, is methylated by aKMT4 in vitro. Mono-methylation of these lysine residues occurs coincidently in the endogenous sisMCM protein purified from the hyperthermophilic Sulfolobus islandicus cells as indicated by mass spectra. The helicase activity of mini-chromosome maintenance (MCM) is stimulated by methylation, particularly at temperatures over 70°C. The methylated MCM shows optimal DNA unwinding activity after heat-treatment between 76 and 82°C, which correlates well with the typical growth temperatures of hyperthermophilic Sulfolobus. After methylation, the half life of MCM helicase is dramatically extended at 80°C. The methylated sites are located on the accessible protein surface, which might modulate the intra- and inter- molecular interactions through changing the hydrophobicity and surface charge. Furthermore, the methylation-mimic mutants of MCM show heat resistance helicase activity comparable to the methylated MCM. These data provide the biochemical evidence that posttranslational modifications such as methylation may enhance kinetic stability of proteins under the elevated growth temperatures of hyperthermophilic archaea. PMID:26617586

  8. Elucidating Proteoform Families from Proteoform Intact-Mass and Lysine-Count Measurements

    PubMed Central

    2016-01-01

    Proteomics is presently dominated by the “bottom-up” strategy, in which proteins are enzymatically digested into peptides for mass spectrometric identification. Although this approach is highly effective at identifying large numbers of proteins present in complex samples, the digestion into peptides renders it impossible to identify the proteoforms from which they were derived. We present here a powerful new strategy for the identification of proteoforms and the elucidation of proteoform families (groups of related proteoforms) from the experimental determination of the accurate proteoform mass and number of lysine residues contained. Accurate proteoform masses are determined by standard LC–MS analysis of undigested protein mixtures in an Orbitrap mass spectrometer, and the lysine count is determined using the NeuCode isotopic tagging method. We demonstrate the approach in analysis of the yeast proteome, revealing 8637 unique proteoforms and 1178 proteoform families. The elucidation of proteoforms and proteoform families afforded here provides an unprecedented new perspective upon proteome complexity and dynamics. PMID:26941048

  9. Structural insights into Parkin substrate lysine targeting from minimal Miro substrates.

    PubMed

    Klosowiak, Julian L; Park, Sungjin; Smith, Kyle P; French, Michael E; Focia, Pamela J; Freymann, Douglas M; Rice, Sarah E

    2016-01-01

    Hereditary Parkinson's disease is commonly caused by mutations in the protein kinase PINK1 or the E3 ubiquitin ligase Parkin, which function together to eliminate damaged mitochondria. PINK1 phosphorylates both Parkin and ubiquitin to stimulate ubiquitination of dozens of proteins on the surface of the outer mitochondrial membrane. However, the mechanisms by which Parkin recognizes specific proteins for modification remain largely unexplored. Here, we show that the C-terminal GTPase (cGTPase) of the Parkin primary substrate human Miro is necessary and sufficient for efficient ubiquitination. We present several new X-ray crystal structures of both human Miro1 and Miro2 that reveal substrate recognition and ubiquitin transfer to be specific to particular protein domains and lysine residues. We also provide evidence that Parkin substrate recognition is functionally separate from substrate modification. Finally, we show that prioritization for modification of a specific lysine sidechain of the cGTPase (K572) within human Miro1 is dependent on both its location and chemical microenvironment. Activation of Parkin by phosphorylation or by binding of pUb is required for prioritization of K572 for modification, suggesting that Parkin activation and acquisition of substrate specificity are coupled. PMID:27605430

  10. Structural insights into Parkin substrate lysine targeting from minimal Miro substrates

    PubMed Central

    Klosowiak, Julian L.; Park, Sungjin; Smith, Kyle P.; French, Michael E.; Focia, Pamela J.; Freymann, Douglas M.; Rice, Sarah E.

    2016-01-01

    Hereditary Parkinson’s disease is commonly caused by mutations in the protein kinase PINK1 or the E3 ubiquitin ligase Parkin, which function together to eliminate damaged mitochondria. PINK1 phosphorylates both Parkin and ubiquitin to stimulate ubiquitination of dozens of proteins on the surface of the outer mitochondrial membrane. However, the mechanisms by which Parkin recognizes specific proteins for modification remain largely unexplored. Here, we show that the C-terminal GTPase (cGTPase) of the Parkin primary substrate human Miro is necessary and sufficient for efficient ubiquitination. We present several new X-ray crystal structures of both human Miro1 and Miro2 that reveal substrate recognition and ubiquitin transfer to be specific to particular protein domains and lysine residues. We also provide evidence that Parkin substrate recognition is functionally separate from substrate modification. Finally, we show that prioritization for modification of a specific lysine sidechain of the cGTPase (K572) within human Miro1 is dependent on both its location and chemical microenvironment. Activation of Parkin by phosphorylation or by binding of pUb is required for prioritization of K572 for modification, suggesting that Parkin activation and acquisition of substrate specificity are coupled. PMID:27605430

  11. Chemoselective small molecules that covalently modify one lysine in a non-enzyme protein in plasma

    SciTech Connect

    Choi, Sungwook; Connelly, Stephen; Reixach, Natàlia; Wilson, Ian A.; Kelly, Jeffery W.

    2010-02-19

    A small molecule that could bind selectively to and then react chemoselectively with a non-enzyme protein in a complex biological fluid, such as blood, could have numerous practical applications. Herein, we report a family of designed stilbenes that selectively and covalently modify the prominent plasma protein transthyretin in preference to more than 4,000 other human plasma proteins. They react chemoselectively with only one of eight lysine {epsilon}-amino groups within transthyretin. The crystal structure confirms the expected binding orientation of the stilbene substructure and the anticipated conjugating amide bond. These covalent transthyretin kinetic stabilizers exhibit superior amyloid inhibition potency compared to their noncovalent counterparts, and they prevent cytotoxicity associated with amyloidogenesis. Though there are a few prodrugs that, upon metabolic activation, react with a cysteine residue inactivating a specific non-enzyme, we are unaware of designed small molecules that react with one lysine {epsilon}-amine within a specific non-enzyme protein in a complex biological fluid.

  12. Lysine11-Linked Polyubiquitination of the AnkB F-Box Effector of Legionella pneumophila

    PubMed Central

    Bruckert, William M.

    2015-01-01

    The fate of the polyubiquitinated protein is determined by the lysine linkages involved in the polymerization of the ubiquitin monomers, which has seven lysine residues (K6, K11, K27, K29, K33, K48, and K63). The translocated AnkB effector of the intravacuolar pathogen Legionella pneumophila is a bona fide F-box protein, which is localized to the cytosolic side of the Legionella-containing vacuole (LCV) and is essential for intravacuolar proliferation within macrophages and amoebae. The F-box domain of AnkB interacts with the host SCF1 E3 ubiquitin ligase that triggers the decoration of the LCV with K48-linked polyubiquitinated proteins that are targeted for proteasomal degradation. Here we report that AnkB becomes rapidly polyubiquitinated within the host cell, and this modification is independent of the F-box domain of AnkB, indicating host-mediated polyubiquitination. We show that the AnkB effector interacts specifically with the host E3 ubiquitin ligase Trim21. Mass spectrometry analyses have shown that AnkB is modified by K11-linked polyubiquitination, which has no effect on its stability. This work shows the first example of K11-linked polyubiquitination of a bacterial effector and its interaction with the host Trim21 ubiquitin ligase. PMID:26483404

  13. Stretch-Induced Helical Conformations in Poly(l-lysine)/Hyaluronic Acid Multilayers.

    PubMed

    Zahouani, Sarah; Chaumont, Alain; Senger, Bernard; Boulmedais, Fouzia; Schaaf, Pierre; Jierry, Loïc; Lavalle, Philippe

    2016-06-22

    We investigate the effect of stretching on the secondary structure of cross-linked poly(l-lysine)/hyaluronic acid (PLL/HA) multilayers. We show that stretching these films induces changes in the secondary structure of PLL chains. Our results suggest that not only α- but also 310-helices might form in the film under stretching. Such 310-helices have never been observed for PLL so far. These changes of the secondary structure of PLL are reversible, i.e., when returning to the nonstretched state one recovers the initial film structure. Using molecular dynamics simulations of chains composed of 20 l-lysine residues (PLL20), we find that these chains never adopt a helical conformation in water. In contrast, when the end-to-end distance of the chains is restrained to values smaller than the mean end-to-end distance of free chains, a distance domain rarely explored by the free chains, helical conformations become accessible. Moreover, the formation of not only α- but also 310-helices is predicted by the simulations. These results suggest that the change of the end-to-end distance of PLL chains in the stretched film is at the origin of the helix formation. PMID:26646202

  14. Identification and Characterization of a Novel Human Methyltransferase Modulating Hsp70 Protein Function through Lysine Methylation*

    PubMed Central

    Jakobsson, Magnus E.; Moen, Anders; Bousset, Luc; Egge-Jacobsen, Wolfgang; Kernstock, Stefan; Melki, Ronald; Falnes, Pål Ø.

    2013-01-01

    Hsp70 proteins constitute an evolutionarily conserved protein family of ATP-dependent molecular chaperones involved in a wide range of biological processes. Mammalian Hsp70 proteins are subject to various post-translational modifications, including methylation, but for most of these, a functional role has not been attributed. In this study, we identified the methyltransferase METTL21A as the enzyme responsible for trimethylation of a conserved lysine residue found in several human Hsp70 (HSPA) proteins. This enzyme, denoted by us as HSPA lysine (K) methyltransferase (HSPA-KMT), was found to catalyze trimethylation of various Hsp70 family members both in vitro and in vivo, and the reaction was stimulated by ATP. Furthermore, we show that HSPA-KMT exclusively methylates 70-kDa proteins in mammalian protein extracts, demonstrating that it is a highly specific enzyme. Finally, we show that trimethylation of HSPA8 (Hsc70) has functional consequences, as it alters the affinity of the chaperone for both the monomeric and fibrillar forms of the Parkinson disease-associated protein α-synuclein. PMID:23921388

  15. A critical assessment of the performance criteria in confirmatory analysis for veterinary drug residue analysis using mass spectrometric detection in selected reaction monitoring mode.

    PubMed

    Berendsen, Bjorn J A; Meijer, Thijs; Wegh, Robin; Mol, Hans G J; Smyth, Wesley G; Armstrong Hewitt, S; van Ginkel, Leen; Nielen, Michel W F

    2016-05-01

    Besides the identification point system to assure adequate set-up of instrumentation, European Commission Decision 2002/657/EC includes performance criteria regarding relative ion abundances in mass spectrometry and chromatographic retention time. In confirmatory analysis, the relative abundance of two product ions, acquired in selected reaction monitoring mode, the ion ratio should be within certain ranges for confirmation of the identity of a substance. The acceptable tolerance of the ion ratio varies with the relative abundance of the two product ions and for retention time, CD 2002/657/EC allows a tolerance of 5%. Because of rapid technical advances in analytical instruments and new approaches applied in the field of contaminant testing in food products (multi-compound and multi-class methods) a critical assessment of these criteria is justified. In this study a large number of representative, though challenging sample extracts were prepared, including muscle, urine, milk and liver, spiked with 100 registered and banned veterinary drugs at levels ranging from 0.5 to 100 µg/kg. These extracts were analysed using SRM mode using different chromatographic conditions and mass spectrometers from different vendors. In the initial study, robust data was collected using four different instrumental set-ups. Based on a unique and highly relevant data set, consisting of over 39 000 data points, the ion ratio and retention time criteria for applicability in confirmatory analysis were assessed. The outcomes were verified based on a collaborative trial including laboratories from all over the world. It was concluded that the ion ratio deviation is not related to the value of the ion ratio, but rather to the intensity of the lowest product ion. Therefore a fixed ion ratio deviation tolerance of 50% (relative) is proposed, which also is applicable for compounds present at sub-ppb levels or having poor ionisation efficiency. Furthermore, it was observed that retention time

  16. An Alternative Strategy for Pan-acetyl-lysine Antibody Generation.

    PubMed

    Kim, Sun-Yee; Sim, Choon Kiat; Zhang, Qiongyi; Tang, Hui; Brunmeir, Reinhard; Pan, Hong; Karnani, Neerja; Han, Weiping; Zhang, Kangling; Xu, Feng

    2016-01-01

    Lysine acetylation is an important post-translational modification in cell signaling. In acetylome studies, a high-quality pan-acetyl-lysine antibody is key to successful enrichment of acetylated peptides for subsequent mass spectrometry analysis. Here we show an alternative method to generate polyclonal pan-acetyl-lysine antibodies using a synthesized random library of acetylated peptides as the antigen. Our antibodies are tested to be specific for acetyl-lysine peptides/proteins via ELISA and dot blot. When pooled, five of our antibodies show broad reactivity to acetyl-lysine peptides, complementing a commercial antibody in terms of peptide coverage. The consensus sequence of peptides bound by our antibody cocktail differs slightly from that of the commercial antibody. Lastly, our antibodies are tested in a proof-of-concept to analyze the acetylome of HEK293 cells. In total we identified 1557 acetylated peptides from 416 proteins. We thus demonstrated that our antibodies are well-qualified for acetylome studies and can complement existing commercial antibodies. PMID:27606599

  17. The 4′lysine in the putative channel lining domain affects desensitization but not the single-channel conductance of recombinant homomeric 5-HT3A receptors

    PubMed Central

    Gunthorpe, Martin J; Peters, John A; Gill, Catherine H; Lambert, Jeremy J; Lummis, Sarah C R

    2000-01-01

    The 5-HT3 receptor is a transmitter-gated ion channel of the Cys-loop superfamily. Uniquely, 5-HT3 receptor subunits (5-HT3A and 5-HT3B) possess a positively charged lysine residue within the putative channel lining M2 domain (4′ position). Using whole cell recording techniques, we examined the role of this residue in receptor function using wild-type (WT) and mutant 5-HT3A receptor subunits of murine origin transiently expressed in human embryonic kidney (HEK 293) cells. WT 5-HT3A receptors mediated rapidly activating currents in response to 5-HT (10–90 % rise time, 103 ms; EC50, 2.34 μm; Hill coefficient, nH, 2.87). The currents rectified inwardly, reversed in sign at a potential of −9 mV and desensitized in the continuous presence of agonist (half-time of desensitization, t1/2, 2.13 s). 5-HT3A receptor subunits in which the 4′lysine was mutated to arginine, glutamine, serine or glycine formed functional receptors. 5-HT EC50 values were approximately 2-fold lower than for WT 5-HT3A receptors, but Hill coefficients, kinetics of current activation, rectification, and reversal potentials were unaltered. Each of the mutants desensitized more slowly than the WT 5-HT3A receptor, with the arginine and glycine mutations exhibiting the greatest effect (5-fold reduction). The rank order of effect was arginine > glycine > serine > glutamine. The single-channel conductance of the WT 5-HT3A receptor, as assessed by fluctuation analysis of macroscopic currents, was 390 fS. A similar value was obtained for the 4′lysine mutant receptors. Thus it appears unlikely that 4′lysine is exposed to the channel lumen. Mutation of residues immediately adjacent to 4′lysine to glutamate or lysine resulted in lack of receptor expression or function. We conclude that 4′lysine does not form part of the channel lining, but may play an important role in 5-HT3 receptor desensitization. PMID:10639097

  18. Seed-specific expression of a lysine-rich protein gene, GhLRP, from cotton significantly increases the lysine content in maize seeds.

    PubMed

    Yue, Jing; Li, Cong; Zhao, Qian; Zhu, Dengyun; Yu, Jingjuan

    2014-01-01

    Maize seed storage proteins are a major source of human and livestock consumption. However, these proteins have poor nutritional value, because they are deficient in lysine and tryptophan. Much research has been done to elevate the lysine content by reducing zein content or regulating the activities of key enzymes in lysine metabolism. Using the naturally lysine-rich protein genes, sb401 and SBgLR, from potato, we previously increased the lysine and protein contents of maize seeds. Here, we examined another natural lysine-rich protein gene, GhLRP, from cotton, which increased the lysine content of transgenic maize seeds at levels varying from 16.2% to 65.0% relative to the wild-type. The total protein content was not distinctly different, except in the six transgenic lines. The lipid and starch levels did not differ substantially in Gossypium hirsutum L. lysine-rich protein (GhLRP) transgenic kernels when compared to wild-type. The agronomic characteristics of all the transgenic maize were also normal. GhLRP is a high-lysine protein candidate gene for increasing the lysine content of maize. This study provided a valuable model system for improving maize lysine content. PMID:24681583

  19. Targeting Lysine Deacetylases (KDACs) in Parasites

    PubMed Central

    Wang, Qi; Rosa, Bruce A.; Nare, Bakela; Powell, Kerrie; Valente, Sergio; Rotili, Dante; Mai, Antonello; Marshall, Garland R.; Mitreva, Makedonka

    2015-01-01

    Due to an increasing problem of drug resistance among almost all parasites species ranging from protists to worms, there is an urgent need to explore new drug targets and their inhibitors to provide new and effective parasitic therapeutics. In this regard, there is growing interest in exploring known drug leads of human epigenetic enzymes as potential starting points to develop novel treatments for parasitic diseases. This approach of repurposing (starting with validated targets and inhibitors) is quite attractive since it has the potential to reduce the expense of drug development and accelerate the process of developing novel drug candidates for parasite control. Lysine deacetylases (KDACs) are among the most studied epigenetic drug targets of humans, and a broad range of small-molecule inhibitors for these enzymes have been reported. In this work, we identify the KDAC protein families in representative species across important classes of parasites, screen a compound library of 23 hydroxamate- or benzamide-based small molecules KDAC inhibitors, and report their activities against a range of parasitic species, including the pathogen of malaria (Plasmodium falciparum), kinetoplastids (Trypanosoma brucei and Leishmania donovani), and nematodes (Brugia malayi, Dirofilaria immitis and Haemonchus contortus). Compound activity against parasites is compared to that observed against the mammalian cell line (L929 mouse fibroblast) in order to determine potential parasite-versus-host selectivity). The compounds showed nanomolar to sub-nanomolar potency against various parasites, and some selectivity was observed within the small panel of compounds tested. The possible binding modes of the active compounds at the different protein target sites within different species were explored by docking to homology models to help guide the discovery of more selective, parasite-specific inhibitors. This current work supports previous studies that explored the use of KDAC inhibitors in

  20. Development of PEGylated Cysteine-Modified Lysine Dendrimers with Multiple Reduced Thiols To Prevent Hepatic Ischemia/Reperfusion Injury.

    PubMed

    Katsumi, Hidemasa; Nishikawa, Makiya; Hirosaki, Rikiya; Okuda, Tatsuya; Kawakami, Shigeru; Yamashita, Fumiyoshi; Hashida, Mitsuru; Sakane, Toshiyasu; Yamamoto, Akira

    2016-08-01

    To inhibit hepatic ischemia/reperfusion injury, we developed polyethylene glycol (PEG) conjugated (PEGylated) cysteine-modified lysine dendrimers with multiple reduced thiols, which function as scavengers of reactive oxygen species (ROS). Second, third, and fourth generation (K2, K3, and K4) highly branched amino acid spherical lysine dendrimers were synthesized, and cysteine (C) was conjugated to the outer layer of these lysine dendrimers to obtain K2C, K3C, and K4C dendrimers. Subsequently, PEG was reacted with the C residues of the dendrimers to obtain PEGylated dendrimers with multiple reduced thiols (K2C-PEG, K3C-PEG, and K4C-PEG). Radiolabeled K4C-PEG ((111)In-K4C-PEG) exhibited prolonged retention in the plasma, whereas (111)In-K2C-PEG and (111)In-K3C-PEG rapidly disappeared from the plasma. K4C-PEG significantly prevented the elevation of plasma alanine aminotransferase (ALT) activity, an index of hepatocyte injury, in a mouse model of hepatic ischemia/reperfusion injury. In contrast, K2C-PEG, K3C-PEG, l-cysteine, and glutathione, the latter two of which are classical reduced thiols, hardly affected the plasma ALT activity. These findings indicate that K4C-PEG with prolonged circulation time is a promising compound to inhibit hepatic ischemia/reperfusion injury. PMID:27336683

  1. Sulfo-NHS-SS-biotin derivatization: a versatile tool for MALDI mass analysis of PTMs in lysine-rich proteins.

    PubMed

    Markoutsa, Stavroula; Bahr, Ute; Papasotiriou, Dimitrios G; Häfner, Ann-Kathrin; Karas, Michael; Sorg, Bernd L

    2014-03-01

    The discovery of PTMs in proteins by MS requires nearly complete sequence coverage of the detected proteolytic peptides. Unfortunately, mass spectrometric analysis of the desired sequence fragments is often impeded due to low ionization efficiency and/or signal suppression in complex samples. When several lysine residues are in close proximity tryptic peptides may be too short for mass analysis. Moreover, modified peptides often appear in low stoichiometry and need to be enriched before analysis. We present here how the use of sulfo-NHS-SS-biotin derivatization of lysine side chain can help to detect PTMs in lysine-rich proteins. This label leads to a mass shift which can be adjusted by reduction of the SS bridge and alkylation with different reagents. Low intensity peptides can be enriched by use of streptavidin beads. Using this method, the functionally relevant protein kinase A phosphorylation site in 5-lipoxygenase was detected for the first time by MS. Additionally, methylation and acetylation could be unambiguously determined in histones. PMID:24449390

  2. KATching-Up on Small Molecule Modulators of Lysine Acetyltransferases.

    PubMed

    Simon, Roman P; Robaa, Dina; Alhalabi, Zayan; Sippl, Wolfgang; Jung, Manfred

    2016-02-25

    The reversible acetylation of lysines is one of the best characterized epigenetic modifications. Its involvement in many key physiological and pathological processes has been documented in numerous studies. Lysine deacetylases (KDACs) and acetyltransferases (KATs) maintain the acetylation equilibrium at histones but also many other proteins. Besides acetylation, also other acyl groups are reversibly installed at the side chain of lysines in proteins. Because of their involvement in disease, KDACs and KATs were proposed to be promising drug targets, and for KDACs, indeed, five inhibitors are now approved for human use. While there is a similar level of evidence for the potential of KATs as drug targets, no inhibitor is in clinical trials. Here, we review the evidence for the diverse roles of KATs in disease pathology, provide an overview of structural features and the available modulators, including those targeting the bromodomains of KATs, and present an outlook. PMID:26701186

  3. Simultaneous detection of lysine metabolites by a single LC-MS/MS method: monitoring lysine degradation in mouse plasma.

    PubMed

    Pena, Izabella A; Marques, Lygia A; Laranjeira, Angelo B A; Yunes, José A; Eberlin, Marcos N; Arruda, Paulo

    2016-01-01

    Detection and quantification of lysine degradation metabolites in plasma is necessary for the diagnosis and follow-up of diseases such as pyridoxine-dependent epilepsy. The principal metabolites involved in the disease are related to the first steps of lysine oxidation, either through the saccharopine or the pipecolate pathways. Currently, there are three different analytical methods used to assess the content of these metabolites in urine and plasma, but they require different sample preparations and analytical equipment. Here, we describe a protocol that calls for a simple sample preparation and uses liquid chromatography tandem mass spectrometry (LC-MS/MS) that allows simultaneous detection and quantification of underivatized l-saccharopine, l-aminoadipic acid, l-pipecolic acid, piperideine-6-carboxylate, l-glutamic acid, and pyridoxal-5-phosphate in plasma samples. To validate the method we analyzed the time course degradation after intraperitoneal injection of l-lysine in C57BL/6/J mice. We observed that the degradation of lysine through the saccharopine pathway reached a maximum within the first 2 h. At this time point there was an increase in the levels of the metabolites saccharopine, aminoadipic acid, and pipecolic acid by 3-, 24- and 3.4-fold, respectively, compared to time zero levels. These metabolites returned to basal levels after 4-6 h. In conclusion, we have developed a LC-MS/MS approach, which allows simultaneous analysis of lysine degradation metabolites without the need for derivatization. PMID:27026869

  4. Reactive lysine content in commercially available pet foods.

    PubMed

    van Rooijen, Charlotte; Bosch, Guido; van der Poel, Antonius F B; Wierenga, Peter A; Alexander, Lucille; Hendriks, Wouter H

    2014-01-01

    The Maillard reaction can occur during processing of pet foods. During this reaction, the ε-amino group of lysine reacts with reducing sugars to become unavailable for metabolism. The aim of the present study was to determine the reactive lysine (RL; the remaining available lysine) to total lysine (TL) ratio of commercial pet foods and to evaluate whether RL levels meet minimal lysine requirements (MLR). Sixty-seven extruded, canned and pelleted commercially available dog and cat foods for growth and maintenance were analysed for proximate nutrient composition, TL and RL. RL was expressed on a metabolisable energy basis and compared with the MLR for maintenance and growth. In dog foods, average RL:TL ratios were 0·87 (se 0·02) for extruded, 0·97 (se 0·02) for canned and 0·85 (se 0·01) for pelleted foods, with the lowest ratio of 0·77 in an extruded diet for growing dogs. In extruded and canned cat foods, the average ratio was 0·91 (se 0·02) and 0·90 (se 0·03), respectively, with the lowest ratio being 0·67 in an extruded diet for growing cats. Variation in the RL:TL ratio between and within processing type indicate that ingredients rather than processing might be the key factor influencing RL content in pet foods. Eight dry foods for growing dogs had RL contents between 96 and 138 % of MLR, indicating that RL has to be between 62 and 104 % digestible to meet the MLR. Considering the variability in RL digestibility, these foods could be at risk of not meeting the MLR for growing dogs. Ingredients and pet foods should be characterised with respect to the RL content and digestibility, to avoid limitations in the lysine supply to growing dogs. PMID:26101604

  5. Lysine fluxes across the jejunal epithelium in lysinuric protein intolerance.

    PubMed

    Desjeux, J F; Simell, R O; Dumontier, A M; Perheentupa, J

    1980-06-01

    Lysinuric protein intolerance (LPI) is one of a group of genetic diseases in which intestinal absorption of the diamino acids lysine, arginine, and ornithine is impaired. In LPI, the clinical symptoms are more severe than in the kindred disorders. The mechanism of lysine absorption was, therefore, investigated in vitro on peroral jejunal biopsy specimens in seven patients with LPI and 27 controls. The lysine concentration ratio between cell compartment and medium was significantly higher in the LPI group (mean+/-SEM, 7.17+/-0.60) than in the controls (5.44+/-0.51). This was also true for the intracellular Na concentration (LPI, 73.6+/-10.8 mM; controls 42.3+/-3.7 mM). The rate of unidirectional influx of lysine across the luminal membrane was Na dependent and was the same in the two groups. In the absence of an electrochemical gradient, net transepithelial lysine secretion was observed in LPI. This was entirely the result of a 60% reduction of the unidirectional flux from mucosa to serosa. Calculation of unidirectional fluxes revealed the most striking difference at the basolateral membrane, where the flux from cells to serosa was reduced by 62% and the corresponding permeability coefficient reduced by 71%. A progressive reduction in short-circuit current appeared in the epithelia of all four patients with LPI tested after addition of 3 mM lysine. Thus, LPI appears to be the first disease in which a genetically determined transport defect has been demonstrated at the basolateral membrane. PMID:6773985

  6. Amino Acid Residues 489–503 of Dihydropyridine Receptor (DHPR) β1a Subunit Are Critical for Structural Communication between the Skeletal Muscle DHPR Complex and Type 1 Ryanodine Receptor*

    PubMed Central

    Eltit, Jose M.; Franzini-Armstrong, Clara; Perez, Claudio F.

    2014-01-01

    The β1a subunit is a cytoplasmic component of the dihydropyridine receptor (DHPR) complex that plays an essential role in skeletal muscle excitation-contraction (EC) coupling. Here we investigate the role of the C-terminal end of this auxiliary subunit in the functional and structural communication between the DHPR and the Ca2+ release channel (RyR1). Progressive truncation of the β1a C terminus showed that deletion of amino acid residues Gln489 to Trp503 resulted in a loss of depolarization-induced Ca2+ release, a severe reduction of L-type Ca2+ currents, and a lack of tetrad formation as evaluated by freeze-fracture analysis. However, deletion of this domain did not affect expression/targeting or density (Qmax) of the DHPR-α1S subunit to the plasma membrane. Within this motif, triple alanine substitution of residues Leu496, Leu500, and Trp503, which are thought to mediate direct β1a-RyR1 interactions, weakened EC coupling but did not replicate the truncated phenotype. Therefore, these data demonstrate that an amino acid segment encompassing sequence 489QVQVLTSLRRNLSFW503 of β1a contains critical determinant(s) for the physical link of DHPR and RyR1, further confirming a direct correspondence between DHPR positioning and DHPR/RyR functional interactions. In addition, our data strongly suggest that the motif Leu496-Leu500-Trp503 within the β1a C-terminal tail plays a nonessential role in the bidirectional DHPR/RyR1 signaling that supports skeletal-type EC coupling. PMID:25384984

  7. Sugar Substrates for l-Lysine Fermentation by Ustilago maydis

    PubMed Central

    Sánchez-Marroquín, A.; Ledezma, M.; Carreño, R.

    1970-01-01

    The extracellular production of l-lysine in media with cane sugar, blackstrap molasses, or clarified sugar-cane juice by a previously obtained mutant of Ustilago maydis was studied. Enzymatically inverted clarified juice (medium J-3) gave 2.9 g of lysine per liter under the following conditions: inoculum, 5%; pH 5.8; temperature, 30 C; KLa in the fermentors, 0.41 mmoles of O2 per liter per min; fermentation time, 72 hr. The concentrate, obtained by direct evaporation and drying of the fermentation broth, could be used as a possible feed supplement because of its amino-acid and vitamin content. PMID:5485081

  8. Sugar substrates for L-lysine fermentation by Ustilago maydis.

    PubMed

    Sánchez-Marroquín, A; Ledezma, M; Carreño, R

    1970-11-01

    The extracellular production of l-lysine in media with cane sugar, blackstrap molasses, or clarified sugar-cane juice by a previously obtained mutant of Ustilago maydis was studied. Enzymatically inverted clarified juice (medium J-3) gave 2.9 g of lysine per liter under the following conditions: inoculum, 5%; pH 5.8; temperature, 30 C; K(La) in the fermentors, 0.41 mmoles of O(2) per liter per min; fermentation time, 72 hr. The concentrate, obtained by direct evaporation and drying of the fermentation broth, could be used as a possible feed supplement because of its amino-acid and vitamin content. PMID:5485081

  9. The pea seedling mitochondrial Nε-lysine acetylome.

    PubMed

    Smith-Hammond, Colin L; Hoyos, Elizabeth; Miernyk, Ján A

    2014-11-01

    Posttranslational lysine acetylation is believed to occur in all taxa and to affect thousands of proteins. In contrast to the hundreds of mitochondrial proteins reported to be lysine-acetylated in non-plant species, only a handful have been reported from the plant taxa previously examined. To investigate whether this reflects a biologically significant difference or merely a peculiarity of the samples thus far examined, we immunoenriched and analyzed acetylated peptides from highly purified pea seedling mitochondria using mass spectrometry. Our results indicate that a multitude of mitochondrial proteins, involved in a variety of processes, are acetylated in pea seedlings. PMID:24780491

  10. Improvement of cell growth and L-lysine production by genetically modified Corynebacterium glutamicum during growth on molasses.

    PubMed

    Xu, Jianzhong; Zhang, Junlan; Guo, Yanfeng; Zai, Yugui; Zhang, Weiguo

    2013-12-01

    Fructose-1,6-bisphosphatase (FBPase) and fructokinase (ScrK) have important roles in regenerating glucose-6-phosphate in the pentose phosphate pathway (PPP), and thus increasing L-lysine production. This article focuses on the development of L-lysine high-producing strains by heterologous expression of FBPase gene fbp and ScrK gene scrK in C. glutamicum lysC (fbr) with molasses as the sole carbon source. Heterologous expression of fbp and scrK lead to a decrease of residual sugar in fermentation broth, and heterologous expression of scrK prevents the fructose efflux. Heterologous expression of fbp and scrK not only increases significantly the activity of corresponding enzymes but also improves cell growth during growth on molasses. FBPase activities are increased tenfold by heterologous expression of fbp, whereas the FBPase activity is only increase fourfold during co-expression of scrK and fbp. Compared with glucose, the DCW of heterologous expression strains are higher on molasses except co-expression of fbp and scrK strain. In addition, heterologous expression of fbp and scrK can strongly increase the L-lysine production with molasses as the sole carbon source. The highest increase (88.4 %) was observed for C. glutamicum lysC (fbr) pDXW-8-fbp-scrK, but the increase was also significant for C. glutamicum lysC (fbr) pDXW-8-fbp (47.2 %) and C. glutamicum lysC (fbr) pDXW-8-scrK (36.8 %). By-products, such as glycerol and dihydroxyacetone, are decreased by heterologous expression of fbp and scrK, whereas trehalose is only slightly increased. The strategy for enhancing L-lysine production by regeneration of glucose-6-phosphate in PPP may provide a reference to enhance the production of other amino acids during growth on molasses or starch. PMID:24029876

  11. A l-Lysine Transporter of High Stereoselectivity of the Amino Acid-Polyamine-Organocation (APC) Superfamily

    PubMed Central

    Kaur, Jagdeep; Olkhova, Elena; Malviya, Viveka Nand; Grell, Ernst; Michel, Hartmut

    2014-01-01

    Membrane proteins of the amino acid-polyamine-organocation (APC) superfamily transport amino acids and amines across membranes and play an important role in the regulation of cellular processes. We report the heterologous production of the LysP-related transporter STM2200 from Salmonella typhimurium in Escherichia coli, its purification, and functional characterization. STM2200 is assumed to be a proton-dependent APC transporter of l-lysine. The functional interaction between basic amino acids and STM2200 was investigated by thermoanalytical methods, i.e. differential scanning and isothermal titration calorimetry. Binding of l-lysine to STM2200 in its solubilized monomer form is entropy-driven. It is characterized by a dissociation constant of 40 μm at pH 5.9 and is highly selective; no evidence was found for the binding of l-arginine, l-ornithine, l-2,4-diaminobutyric acid, and l-alanine. d-Lysine is bound 45 times more weakly than its l-chiral form. We thus postulate that STM2200 functions as a specific transport protein. Based on the crystal structure of ApcT (Shaffer, P. L., Goehring, A., Shankaranarayanan, A., and Gouaux, E. (2009) Science 325, 1010–1014), a proton-dependent amino acid transporter of the APC superfamily, a homology model of STM2200 was created. Docking studies allowed identification of possible ligand binding sites. The resulting predictions indicated that Glu-222 and Arg-395 of STM2200 are markedly involved in ligand binding, whereas Lys-163 is suggested to be of structural and functional relevance. Selected variants of STM2200 where these three amino acid residues were substituted using single site-directed mutagenesis showed no evidence for l-lysine binding by isothermal titration calorimetry, which confirmed the predictions. Molecular aspects of the observed ligand specificity are discussed. PMID:24257746

  12. Superoxide reductase from Desulfoarculus baarsii: reaction mechanism and role of glutamate 47 and lysine 48 in catalysis.

    PubMed

    Lombard, M; Houée-Levin, C; Touati, D; Fontecave, M; Nivière, V

    2001-04-24

    Superoxide reductase (SOR) is a small metalloenzyme that catalyzes reduction of O(2)(*)(-) to H(2)O(2) and thus provides an antioxidant mechanism against superoxide radicals. Its active site contains an unusual mononuclear ferrous center, which is very efficient during electron transfer to O(2)(*)(-) [Lombard, M., Fontecave, M., Touati, D., and Nivière, V. (2000) J. Biol. Chem. 275, 115-121]. The reaction of the enzyme from Desulfoarculus baarsii with superoxide was studied by pulse radiolysis methods. The first step is an extremely fast bimolecular reaction of superoxide reductase with superoxide, with a rate constant of (1.1 +/- 0.3) x 10(9) M(-1) s(-1). A first intermediate is formed which is converted to a second one at a much slower rate constant of 500 +/- 50 s(-1). Decay of the second intermediate occurs with a rate constant of 25 +/- 5 s(-1). These intermediates are suggested to be iron-superoxide and iron-peroxide species. Furthermore, the role of glutamate 47 and lysine 48, which are the closest charged residues to the vacant sixth iron coordination site, has been investigated by site-directed mutagenesis. Mutation of glutamate 47 into alanine has no effect on the rates of the reaction. On the contrary, mutation of lysine 48 into an isoleucine led to a 20-30-fold decrease of the rate constant of the bimolecular reaction, suggesting that lysine 48 plays an important role during guiding and binding of superoxide to the iron center II. In addition, we report that expression of the lysine 48 sor mutant gene hardly restored to a superoxide dismutase-deficient Escherichia coli mutant the ability to grow under aerobic conditions. PMID:11305919

  13. Structural aspects of the solvation shell of lysine and acetylated lysine: A Car-Parrinello and classical molecular dynamics investigation

    NASA Astrophysics Data System (ADS)

    Carnevale, V.; Raugei, S.

    2009-12-01

    Lysine acetylation is a post-translational modification, which modulates the affinity of protein-protein and/or protein-DNA complexes. Its crucial role as a switch in signaling pathways highlights the relevance of charged chemical groups in determining the interactions between water and biomolecules. A great effort has been recently devoted to assess the reliability of classical molecular dynamics simulations in describing the solvation properties of charged moieties. In the spirit of these investigations, we performed classical and Car-Parrinello molecular dynamics simulations on lysine and acetylated-lysine in aqueous solution. A comparative analysis between the two computational schemes is presented with a focus on the first solvation shell of the charged groups. An accurate structural analysis unveils subtle, yet statistically significant, differences which are discussed in connection to the significant electronic density charge transfer occurring between the solute and the surrounding water molecules.

  14. Structural aspects of the solvation shell of lysine and acetylated lysine: A Car-Parrinello and classical molecular dynamics investigation

    SciTech Connect

    Carnevale, V.; Raugei, S.

    2009-12-14

    Lysine acetylation is a post-translational modification, which modulates the affinity of protein-protein and/or protein-DNA complexes. Its crucial role as a switch in signaling pathways highlights the relevance of charged chemical groups in determining the interactions between water and biomolecules. A great effort has been recently devoted to assess the reliability of classical molecular dynamics simulations in describing the solvation properties of charged moieties. In the spirit of these investigations, we performed classical and Car-Parrinello molecular dynamics simulations on lysine and acetylated-lysine in aqueous solution. A comparative analysis between the two computational schemes is presented with a focus on the first solvation shell of the charged groups. An accurate structural analysis unveils subtle, yet statistically significant, differences which are discussed in connection to the significant electronic density charge transfer occurring between the solute and the surrounding water molecules.

  15. Strictly Conserved Lysine of Prolyl-tRNA Synthetase Editing Domain Facilitates Binding and Positioning of Misacylated tRNAPro

    PubMed Central

    2015-01-01

    To ensure high fidelity in translation, many aminoacyl-tRNA synthetases, enzymes responsible for attaching specific amino acids to cognate tRNAs, require proof-reading mechanisms. Most bacterial prolyl-tRNA synthetases (ProRSs) misactivate alanine and employ a post-transfer editing mechanism to hydrolyze Ala-tRNAPro. This reaction occurs in a second catalytic site (INS) that is distinct from the synthetic active site. The 2′-OH of misacylated tRNAPro and several conserved residues in the Escherichia coli ProRS INS domain are directly involved in Ala-tRNAPro deacylation. Although mutation of the strictly conserved lysine 279 (K279) results in nearly complete loss of post-transfer editing activity, this residue does not directly participate in Ala-tRNAPro hydrolysis. We hypothesized that the role of K279 is to bind the phosphate backbone of the acceptor stem of misacylated tRNAPro and position it in the editing active site. To test this hypothesis, we carried out pKa, charge neutralization, and free-energy of binding calculations. Site-directed mutagenesis and kinetic studies were performed to verify the computational results. The calculations revealed a considerably higher pKa of K279 compared to an isolated lysine and showed that the protonated state of K279 is stabilized by the neighboring acidic residue. However, substitution of this acidic residue with a positively charged residue leads to a significant increase in Ala-tRNAPro hydrolysis, suggesting that enhancement in positive charge density in the vicinity of K279 favors tRNA binding. A charge-swapping experiment and free energy of binding calculations support the conclusion that the positive charge at position 279 is absolutely necessary for tRNA binding in the editing active site. PMID:24450765

  16. Crop residues

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Crop residues [e.g., corn (Zea mays) stover and small grain straw] are sometimes excluded when discussing cellulosic energy crops per se, but because of the vast area upon which they are grown and their current role in the development of cellulosic energy systems. This chapter focuses on current cor...

  17. Chemoproteomic Profiling of Lysine Acetyltransferases Highlights an Expanded Landscape of Catalytic Acetylation

    PubMed Central

    2015-01-01

    Lysine acetyltransferases (KATs) play a critical role in the regulation of gene expression, metabolism, and other key cellular functions. One shortcoming of traditional KAT assays is their inability to study KAT activity in complex settings, a limitation that hinders efforts at KAT discovery, characterization, and inhibitor development. To address this challenge, here we describe a suite of cofactor-based affinity probes capable of profiling KAT activity in biological contexts. Conversion of KAT bisubstrate inhibitors to clickable photoaffinity probes enables the selective covalent labeling of three phylogenetically distinct families of KAT enzymes. Cofactor-based affinity probes report on KAT activity in cell lysates, where KATs exist as multiprotein complexes. Chemical affinity purification and unbiased LC–MS/MS profiling highlights an expanded landscape of orphan lysine acetyltransferases present in the human genome and provides insight into the global selectivity and sensitivity of CoA-based proteomic probes that will guide future applications. Chemoproteomic profiling provides a powerful method to study the molecular interactions of KATs in native contexts and will aid investigations into the role of KATs in cell state and disease. PMID:24836640

  18. The Lysine Acetyltransferase Activator Brpf1 Governs Dentate Gyrus Development through Neural Stem Cells and Progenitors

    PubMed Central

    You, Linya; Yan, Kezhi; Zhou, Jinfeng; Zhao, Hong; Bertos, Nicholas R.; Park, Morag; Wang, Edwin; Yang, Xiang-Jiao

    2015-01-01

    Lysine acetylation has recently emerged as an important post-translational modification in diverse organisms, but relatively little is known about its roles in mammalian development and stem cells. Bromodomain- and PHD finger-containing protein 1 (BRPF1) is a multidomain histone binder and a master activator of three lysine acetyltransferases, MOZ, MORF and HBO1, which are also known as KAT6A, KAT6B and KAT7, respectively. While the MOZ and MORF genes are rearranged in leukemia, the MORF gene is also mutated in prostate and other cancers and in four genetic disorders with intellectual disability. Here we show that forebrain-specific inactivation of the mouse Brpf1 gene causes hypoplasia in the dentate gyrus, including underdevelopment of the suprapyramidal blade and complete loss of the infrapyramidal blade. We trace the developmental origin to compromised Sox2+ neural stem cells and Tbr2+ intermediate neuronal progenitors. We further demonstrate that Brpf1 loss deregulates neuronal migration, cell cycle progression and transcriptional control, thereby causing abnormal morphogenesis of the hippocampus. These results link histone binding and acetylation control to hippocampus development and identify an important epigenetic regulator for patterning the dentate gyrus, a brain structure critical for learning, memory and adult neurogenesis. PMID:25757017

  19. The lysine acetyltransferase activator Brpf1 governs dentate gyrus development through neural stem cells and progenitors.

    PubMed

    You, Linya; Yan, Kezhi; Zou, Jinfeng; Zhou, Jinfeng; Zhao, Hong; Bertos, Nicholas R; Park, Morag; Wang, Edwin; Yang, Xiang-Jiao

    2015-03-01

    Lysine acetylation has recently emerged as an important post-translational modification in diverse organisms, but relatively little is known about its roles in mammalian development and stem cells. Bromodomain- and PHD finger-containing protein 1 (BRPF1) is a multidomain histone binder and a master activator of three lysine acetyltransferases, MOZ, MORF and HBO1, which are also known as KAT6A, KAT6B and KAT7, respectively. While the MOZ and MORF genes are rearranged in leukemia, the MORF gene is also mutated in prostate and other cancers and in four genetic disorders with intellectual disability. Here we show that forebrain-specific inactivation of the mouse Brpf1 gene causes hypoplasia in the dentate gyrus, including underdevelopment of the suprapyramidal blade and complete loss of the infrapyramidal blade. We trace the developmental origin to compromised Sox2+ neural stem cells and Tbr2+ intermediate neuronal progenitors. We further demonstrate that Brpf1 loss deregulates neuronal migration, cell cycle progression and transcriptional control, thereby causing abnormal morphogenesis of the hippocampus. These results link histone binding and acetylation control to hippocampus development and identify an important epigenetic regulator for patterning the dentate gyrus, a brain structure critical for learning, memory and adult neurogenesis. PMID:25757017

  20. Bovine NK-lysin: Copy number variation and functional diversification

    PubMed Central

    Chen, Junfeng; Huddleston, John; Buckley, Reuben M.; Malig, Maika; Lawhon, Sara D.; Skow, Loren C.; Lee, Mi Ok; Eichler, Evan E.; Andersson, Leif; Womack, James E.

    2015-01-01

    NK-lysin is an antimicrobial peptide and effector protein in the host innate immune system. It is coded by a single gene in humans and most other mammalian species. In this study, we provide evidence for the existence of four NK-lysin genes in a repetitive region on cattle chromosome 11. The NK2A, NK2B, and NK2C genes are tandemly arrayed as three copies in ∼30–35-kb segments, located 41.8 kb upstream of NK1. All four genes are functional, albeit with differential tissue expression. NK1, NK2A, and NK2B exhibited the highest expression in intestine Peyer’s patch, whereas NK2C was expressed almost exclusively in lung. The four peptide products were synthesized ex vivo, and their antimicrobial effects against both Gram-positive and Gram-negative bacteria were confirmed with a bacteria-killing assay. Transmission electron microcopy indicated that bovine NK-lysins exhibited their antimicrobial activities by lytic action in the cell membranes. In summary, the single NK-lysin gene in other mammals has expanded to a four-member gene family by tandem duplications in cattle; all four genes are transcribed, and the synthetic peptides corresponding to the core regions are biologically active and likely contribute to innate immunity in ruminants. PMID:26668394

  1. Bovine NK-lysin: Copy number variation and functional diversification.

    PubMed

    Chen, Junfeng; Huddleston, John; Buckley, Reuben M; Malig, Maika; Lawhon, Sara D; Skow, Loren C; Lee, Mi Ok; Eichler, Evan E; Andersson, Leif; Womack, James E

    2015-12-29

    NK-lysin is an antimicrobial peptide and effector protein in the host innate immune system. It is coded by a single gene in humans and most other mammalian species. In this study, we provide evidence for the existence of four NK-lysin genes in a repetitive region on cattle chromosome 11. The NK2A, NK2B, and NK2C genes are tandemly arrayed as three copies in ∼30-35-kb segments, located 41.8 kb upstream of NK1. All four genes are functional, albeit with differential tissue expression. NK1, NK2A, and NK2B exhibited the highest expression in intestine Peyer's patch, whereas NK2C was expressed almost exclusively in lung. The four peptide products were synthesized ex vivo, and their antimicrobial effects against both Gram-positive and Gram-negative bacteria were confirmed with a bacteria-killing assay. Transmission electron microcopy indicated that bovine NK-lysins exhibited their antimicrobial activities by lytic action in the cell membranes. In summary, the single NK-lysin gene in other mammals has expanded to a four-member gene family by tandem duplications in cattle; all four genes are transcribed, and the synthetic peptides corresponding to the core regions are biologically active and likely contribute to innate immunity in ruminants. PMID:26668394

  2. Identification and functional characterization of lysine methyltransferases of Entamoeba histolytica.

    PubMed

    Borbolla-Vázquez, Jessica; Orozco, Esther; Medina-Gómez, Christian; Martínez-Higuera, Aarón; Javier-Reyna, Rosario; Chávez, Bibiana; Betanzos, Abigail; Rodríguez, Mario A

    2016-07-01

    Lysine methylation of histones, a posttranslational modification catalyzed by lysine methyltransferases (HKMTs), plays an important role in the epigenetic regulation of transcription. Lysine methylation of non-histone proteins also impacts the biological function of proteins. Previously it has been shown that lysine methylation of histones of Entamoeba histolytica, the protozoan parasite that infects 50 million people worldwide each year and causing up to 100,000 deaths annually, is implicated in the epigenetic machinery of this microorganism. However, the identification and characterization of HKMTs in this parasite had not yet been determined. In this work we identified four HKMTs in E. histolytica (EhHKMT1 to EhHKMT4) that are expressed by trophozoites. Enzymatic assays indicated that all of them are able to transfer methyl groups to commercial histones. EhHKMT1, EhHKMT2 and EhHKMT4 were detected in nucleus and cytoplasm of trophozoites. In addition EhHKMT2 and EhHKMT4 were located in vesicles containing ingested cells during phagocytosis, and they co-immunoprecipitated with EhADH, a protein involved in the phagocytosis of this parasite. Results suggest that E. histolytica uses its HKMTs to regulate transcription by epigenetic mechanisms, and at least two of them could also be implicated in methylation of proteins that participate in phagocytosis. PMID:27062489

  3. Post-translational Serine/Threonine Phosphorylation and Lysine Acetylation: A Novel Regulatory Aspect of the Global Nitrogen Response Regulator GlnR in S. coelicolor M145

    PubMed Central

    Amin, Rafat; Franz-Wachtel, Mirita; Tiffert, Yvonne; Heberer, Martin; Meky, Mohamed; Ahmed, Yousra; Matthews, Arne; Krysenko, Sergii; Jakobi, Marco; Hinder, Markus; Moore, Jane; Okoniewski, Nicole; Maček, Boris; Wohlleben, Wolfgang; Bera, Agnieszka

    2016-01-01

    Soil-dwelling Streptomyces bacteria such as S.coelicolor have to constantly adapt to the nitrogen (N) availability in their habitat. Thus, strict transcriptional and post-translational control of the N-assimilation is fundamental for survival of this species. GlnR is a global response regulator that controls transcription of the genes related to the N-assimilation in S. coelicolor and other members of the Actinomycetales. GlnR represents an atypical orphan response regulator that is not activated by the phosphorylation of the conserved aspartate residue (Asp 50). We have applied transcriptional analysis, LC-MS/MS analysis and electrophoretic mobility shift assays (EMSAs) to understand the regulation of GlnR in S. coelicolor M145. The expression of glnR and GlnR-target genes was revisited under four different N-defined conditions and a complex N-rich condition. Although, the expression of selected GlnR-target genes was strongly responsive to changing N-concentrations, the glnR expression itself was independent of the N-availability. Using LC-MS/MSanalysis we demonstrated that GlnR was post-translationally modified. The post-translational modifications of GlnR comprise phosphorylation of the serine/threonine residues and acetylation of lysine residues. In the complex N-rich medium GlnR was phosphorylated on six serine/threonine residues and acetylated on one lysine residue. Under defined N-excess conditions only two phosphorylated residues were detected whereas under defined N-limiting conditions no phosphorylation was observed. GlnR phosphorylation is thus clearly correlated with N-rich conditions. Furthermore, GlnR was acetylated on four lysine residues independently of the N-concentration in the defined media and on only one lysine residue in the complex N-rich medium. Using EMSAs we demonstrated that phosphorylation inhibited the binding of GlnR to its targets genes, whereas acetylation had little influence on the formation of GlnR-DNA complex. This study clearly

  4. Post-translational Serine/Threonine Phosphorylation and Lysine Acetylation: A Novel Regulatory Aspect of the Global Nitrogen Response Regulator GlnR in S. coelicolor M145.

    PubMed

    Amin, Rafat; Franz-Wachtel, Mirita; Tiffert, Yvonne; Heberer, Martin; Meky, Mohamed; Ahmed, Yousra; Matthews, Arne; Krysenko, Sergii; Jakobi, Marco; Hinder, Markus; Moore, Jane; Okoniewski, Nicole; Maček, Boris; Wohlleben, Wolfgang; Bera, Agnieszka

    2016-01-01

    Soil-dwelling Streptomyces bacteria such as S.coelicolor have to constantly adapt to the nitrogen (N) availability in their habitat. Thus, strict transcriptional and post-translational control of the N-assimilation is fundamental for survival of this species. GlnR is a global response regulator that controls transcription of the genes related to the N-assimilation in S. coelicolor and other members of the Actinomycetales. GlnR represents an atypical orphan response regulator that is not activated by the phosphorylation of the conserved aspartate residue (Asp 50). We have applied transcriptional analysis, LC-MS/MS analysis and electrophoretic mobility shift assays (EMSAs) to understand the regulation of GlnR in S. coelicolor M145. The expression of glnR and GlnR-target genes was revisited under four different N-defined conditions and a complex N-rich condition. Although, the expression of selected GlnR-target genes was strongly responsive to changing N-concentrations, the glnR expression itself was independent of the N-availability. Using LC-MS/MSanalysis we demonstrated that GlnR was post-translationally modified. The post-translational modifications of GlnR comprise phosphorylation of the serine/threonine residues and acetylation of lysine residues. In the complex N-rich medium GlnR was phosphorylated on six serine/threonine residues and acetylated on one lysine residue. Under defined N-excess conditions only two phosphorylated residues were detected whereas under defined N-limiting conditions no phosphorylation was observed. GlnR phosphorylation is thus clearly correlated with N-rich conditions. Furthermore, GlnR was acetylated on four lysine residues independently of the N-concentration in the defined media and on only one lysine residue in the complex N-rich medium. Using EMSAs we demonstrated that phosphorylation inhibited the binding of GlnR to its targets genes, whereas acetylation had little influence on the formation of GlnR-DNA complex. This study clearly

  5. A de novo NADPH generation pathway for improving lysine production of Corynebacterium glutamicum by rational design of the coenzyme specificity of glyceraldehyde 3-phosphate dehydrogenase.

    PubMed

    Bommareddy, Rajesh Reddy; Chen, Zhen; Rappert, Sugima; Zeng, An-Ping

    2014-09-01

    Engineering the cofactor availability is a common strategy of metabolic engineering to improve the production of many industrially important compounds. In this work, a de novo NADPH generation pathway is proposed by altering the coenzyme specificity of a native NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to NADP, which consequently has the potential to produce additional NADPH in the glycolytic pathway. Specifically, the coenzyme specificity of GAPDH of Corynebacterium glutamicum is systematically manipulated by rational protein design and the effect of the manipulation for cellular metabolism and lysine production is evaluated. By a combinatorial modification of four key residues within the coenzyme binding sites, different GAPDH mutants with varied coenzyme specificity were constructed. While increasing the catalytic efficiency of GAPDH towards NADP enhanced lysine production in all of the tested mutants, the most significant improvement of lysine production (~60%) was achieved with the mutant showing similar preference towards both NAD and NADP. Metabolic flux analysis with (13)C isotope studies confirmed that there was no significant change of flux towards the pentose phosphate pathway and the increased lysine yield was mainly attributed to the NADPH generated by the mutated GAPDH. The present study highlights the importance of protein engineering as a key strategy in de novo pathway design and overproduction of desired products. PMID:24953302

  6. Lysine Biosynthesis in Barley (Hordeum vulgare L.) 1

    PubMed Central

    Møller, Birger Lindberg

    1974-01-01

    Lysine biosynthesis in seedlings of barley (Hordeum vulgare L. var. Emir) was studied by direct injection of the following precursors into the endosperm of the seedlings: acetate-1-14C; acetate-2-14C; pyruvate-1-14C; pyruvate-2-14C; pyruvate-3-14C; alanine-1-14C; aspartic acid-1-14C; aspartic acid-2-14C; aspartic acid-3-14C; aspartic acid-4-14C; α-aminoadipic acid-1-14C; and α, ε-diaminopimelic acid-1-(7)-14C. The distribution of activity in the individual carbon atoms of lysine in the different biosynthetic experiments was determined by chemical degradation. The incorporation percentages and labeling patterns obtained are in agreement with the occurrence of the diaminopimelic acid pathway. The results do not fit the incorporation percentages and labeling patterns expected if the α-aminoadipic acid pathway was operating. However, the results show that barley seedlings are able to convert a small part of the α-aminoadipic acid administered directly to lysine. The labeling pattern of lysine was found to be symmetrical around carbon 4. This indicates that the biosynthetic pathway proceeds via a symmetrical intermediate like ll-α, ε-diaminopimelic acid, or includes compounds as 2, 3-dihydrodipicolinic acid or Δ1-piperideine-2, 6-dicarboxylic acid which probably isomerise with concomitant lack of asymmetry in the labeling. The percentages of incorporation show that both the mesoand ll-forms of α, ε-diaminopimelic acid are metabolically convertible to lysine in seedlings of barley. PMID:16658942

  7. Correlation of carnitine levels to methionine and lysine intake.

    PubMed

    Krajcovicová-Kudlácková, M; Simoncic, R; Béderová, A; Babinská, K; Béder, I

    2000-01-01

    Plasma carnitine levels were measured in two alternative nutrition groups--strict vegetarians (vegans) and lactoovovegetarians (vegetarians consuming limited amounts of animal products such as milk products and eggs). The results were compared to an average sample of probands on mixed nutrition (omnivores). Carnitine levels were correlated with the intake of essential amino acids, methionine and lysine (as substrates of its endogenous synthesis), since the intake of carnitine in food is negligible in the alternative nutrition groups (the highest carnitine content is in meat, lower is in milk products, while fruit, cereals and vegetables contain low or no carnitine at all). An average carnitine level in vegans was significantly reduced with hypocarnitinemia present in 52.9% of probands. Similarly, the intake of methionine and lysine was significantly lower in this group due to the exclusive consumption of plant proteins with reduced content of these amino acids. Carnitine level in lactoovovegetarians was also significantly reduced, but the incidence of values below 30 micromol/l was lower than in vegans representing 17.8% vs. 3.3% in omnivores. Intake of methionine and lysine was also significantly reduced in this group, but still higher compared to vegans (73% of protein intake covered by plant proteins). Significant positive correlation of carnitine levels with methionine and lysine intake in alternative nutrition groups indicates that a significant portion of carnitine requirement is covered by endogenous synthesis. Approximately two thirds of carnitine requirement in omnivores comes from exogenous sources. The results demonstrate the risks of alternative nutrition with respect to the intake of essential amino acids, methionine and lysine, and with respect to the intake and biosynthesis of carnitine. PMID:11043928

  8. Possible Evidence of Amide Bond Formation Between Sinapinic Acid and Lysine-Containing Bacterial Proteins by Matrix-Assisted Laser Desorption/Ionization (MALDI) at 355 nm

    NASA Astrophysics Data System (ADS)

    Fagerquist, Clifton K.; Sultan, Omar; Carter, Michelle Q.

    2012-12-01

    We previously reported the apparent formation of matrix adducts of 3,5-dimethoxy-4-hydroxy-cinnamic acid (sinapinic acid or SA) via covalent attachment to disulfide bond-containing proteins (HdeA, Hde, and YbgS) from bacterial cell lysates ionized by matrix-assisted laser desorption/ionization (MALDI) time-of-flight-time-of-flight tandem mass spectrometry (TOF-TOF-MS/MS) and post-source decay (PSD). We also reported the absence of adduct formation when using α-cyano-4-hydroxycinnamic acid (CHCA) matrix. Further mass spectrometric analysis of disulfide-intact and disulfide-reduced over-expressed HdeA and HdeB proteins from lysates of gene-inserted E. coli plasmids suggests covalent attachment of SA occurs not at cysteine residues but at lysine residues. In this revised hypothesis, the attachment of SA is preceded by formation of a solid phase ammonium carboxylate salt between SA and accessible lysine residues of the protein during sample preparation under acidic conditions. Laser irradiation at 355 nm of the dried sample spot results in equilibrium retrogradation followed by nucleophilic attack by the amine group of lysine at the carbonyl group of SA and subsequent amide bond formation and loss of water. The absence of CHCA adducts suggests that the electron-withdrawing effect of the α-cyano group of this matrix may inhibit salt formation and/or amide bond formation. This revised hypothesis is supported by dissociative loss of SA (-224 Da) and the amide-bound SA (-206 Da) from SA-adducted HdeA and HdeB ions by MS/MS (PSD). It is proposed that cleavage of the amide-bound SA from the lysine side-chain occurs via rearrangement involving a pentacyclic transition state followed by hydrogen abstraction/migration and loss of 3-(4-hydroxy-3,5-dimethoxyphenyl)prop-2-ynal (-206 Da).

  9. In vitro degradation of lysine by ruminal fluid-based fermentations and by Fusobacterium necrophorum.

    PubMed

    Elwakeel, E A; Amachawadi, R G; Nour, A M; Nasser, M E A; Nagaraja, T G; Titgemeyer, E C

    2013-01-01

    The objective of these studies was to characterize some factors affecting lysine degradation by mixed ruminal bacteria and by ruminal Fusobacterium necrophorum. Mixed ruminal bacteria degraded lysine, and addition of pure cultures of F. necrophorum did not increase lysine degradation. Addition of acetic or propionic acid strikingly reduced NH(3) production from lysine by mixed ruminal bacteria at pH 6, but not at pH 7. Although typical ruminal environments with acidic pH and normal concentrations of volatile fatty acids might inhibit lysine degradation by F. necrophorum, ruminal fluid contained enough bacteria with a lysine-degrading capacity to ferment 50 mM lysine in vitro. Of 7 strains of ruminal F. necrophorum tested, all grew on both lactate and lysine as the primary energy source. Both subspecies of ruminal F. necrophorum (necrophorum and funduliforme) used lysine as a primary C and energy source. Lysine and glutamic acid were effectively fermented by F. necrophorum, but alanine and tryptophan were not, and histidine and methionine were fermented only to a minor extent. The end products of lactate fermentation by F. necrophorum were propionate and acetate, and those of lysine degradation were butyrate and acetate. Fermentation of glutamic acid by F. necrophorum yielded acetate and butyrate in a ratio near to 2:1. The minimum inhibitory concentration of tylosin for F. necrophorum was not dependent on whether bacteria were grown with lactate or lysine, but F. necrophorum was more susceptible to monensin when grown on lysine than on lactate. Although F. necrophorum is generally resistant to monensin, the ionophore may reduce lysine degradation by F. necrophorum in the rumen. The essential oil components limonene, at 20 or 100 μg/mL, and thymol, at 100 μg/mL, inhibited F. necrophorum growth, whereas eugenol, guaiacol, and vanillin had no effect. Our findings may lead to ways to minimize ruminal lysine degradation and thus increase its availability to the animal

  10. Single residue substitutions that change the gating properties of a mechanosensitive channel in Escherichia coli

    NASA Technical Reports Server (NTRS)

    Blount, P.; Sukharev, S. I.; Schroeder, M. J.; Nagle, S. K.; Kung, C.

    1996-01-01

    MscL is a channel that opens a large pore in the Escherichia coli cytoplasmic membrane in response to mechanical stress. Previously, we highly enriched the MscL protein by using patch clamp as a functional assay and cloned the corresponding gene. The predicted protein contains a largely hydrophobic core spanning two-thirds of the molecule and a more hydrophilic carboxyl terminal tail. Because MscL had no homology to characterized proteins, it was impossible to predict functional regions of the protein by simple inspection. Here, by mutagenesis, we have searched for functionally important regions of this molecule. We show that a short deletion from the amino terminus (3 amino acids), and a larger deletion of 27 amino acids from the carboxyl terminus of this protein, had little if any effect in channel properties. We have thus narrowed the search of the core mechanosensitive mechanism to 106 residues of this 136-amino acid protein. In contrast, single residue substitutions of a lysine in the putative first transmembrane domain or a glutamine in the periplasmic loop caused pronounced shifts in the mechano-sensitivity curves and/or large changes in the kinetics of channel gating, suggesting that the conformational structure in these regions is critical for normal mechanosensitive channel gating.

  11. From a pro-apoptotic peptide to a lytic peptide: One single residue mutation.

    PubMed

    Zhou, Xi-Rui; Zhang, Qiang; Tian, Xi-Bo; Cao, Yi-Meng; Liu, Zhu-Qing; Fan, Ruru; Ding, Xiu-Fang; Zhu, Zhentai; Chen, Long; Luo, Shi-Zhong

    2016-08-01

    Further discovery and design of new anticancer peptides are important for the development of anticancer therapeutics, and study on the detailed acting mechanism and structure-function relationship of peptides is critical for anticancer peptide design and application. In this study, a novel anticancer peptide ZXR-1 (FKIGGFIKKLWRSKLA) derived from a known anticancer peptide mauriporin was developed, and a mutant ZXR-2 (FKIGGFIKKLWRSLLA) with only one residue difference at the 14th position (Lys→Leu) was also engineered. Replacement of the lysine with leucine made ZXR-2 more potent than ZXR-1 in general. Even with only one residue mutation, the two peptides displayed distinct anticancer modes of action. ZXR-1 could translocate into cells, target on the mitochondria and induce cell apoptosis, while ZXR-2 directly targeted on the cell membranes and caused membrane lysis. The variance in their acting mechanisms might be due to the different amphipathicity and positive charge distribution. In addition, the two Ile-Leu pairs (3-10 and 7-14) in ZXR-2 might also play a role in improving its cytotoxicity. Further study on the structure-function relationship of the two peptides may be beneficial for the design of novel anticancer peptides and peptide based therapeutics. PMID:27207743

  12. Single residue substitutions that change the gating properties of a mechanosensitive channel in Escherichia coli.

    PubMed Central

    Blount, P; Sukharev, S I; Schroeder, M J; Nagle, S K; Kung, C

    1996-01-01

    MscL is a channel that opens a large pore in the Escherichia coli cytoplasmic membrane in response to mechanical stress. Previously, we highly enriched the MscL protein by using patch clamp as a functional assay and cloned the corresponding gene. The predicted protein contains a largely hydrophobic core spanning two-thirds of the molecule and a more hydrophilic carboxyl terminal tail. Because MscL had no homology to characterized proteins, it was impossible to predict functional regions of the protein by simple inspection. Here, by mutagenesis, we have searched for functionally important regions of this molecule. We show that a short deletion from the amino terminus (3 amino acids), and a larger deletion of 27 amino acids from the carboxyl terminus of this protein, had little if any effect in channel properties. We have thus narrowed the search of the core mechanosensitive mechanism to 106 residues of this 136-amino acid protein. In contrast, single residue substitutions of a lysine in the putative first transmembrane domain or a glutamine in the periplasmic loop caused pronounced shifts in the mechano-sensitivity curves and/or large changes in the kinetics of channel gating, suggesting that the conformational structure in these regions is critical for normal mechanosensitive channel gating. Images Fig. 3 PMID:8876191

  13. Site-specific Labeling of a Protein Lysine Residue By Novel Kinetic Labeling Combinatorial Libraries

    PubMed Central

    Krantz, Allen; Hanel, Arthur M; Strug, Ivona; Wilczynski, Andrzej; Wolff, Jeremy J; Huang, Wolin; Huang, Linda H; Settineri, Tina; Holmes, Darren L; Hardy, Margaret C; Bridon, Dominique P

    2014-01-01

    The first example of a kinetic labeling library designed to enable the discovery of affinity labels is presented. Each library component (1) consists of a variable peptidyl component linked to a biotinyl moiety by a 4-mercaptobenzoyl linker in thioester format. We demonstrate that an affinity label can be uncovered by measuring reaction rates between library pools and the protein target, human serum albumin (HSA) and identifying significant outliers. By choosing peptide functionality compatible with a potentially reactive thioester labeling entity, libraries can be screened in pools. It is noteworthy that a limited subset of amino acids (R, S, E, F, Y, l, M, W, and Q) that compose the affinity moiety is sufficient to produce rate variances that guide the discovery process. After two rounds of deconvolution, J-FLYEE-NH2 (7-E) emerges as a bona fide affinity label of HSA. Unlike known affinity labels, the affinity moiety is not retained in the protein product, but is extruded upon acylation of the protein. This feature affords a method of introducing various payloads, without extraneous elements, onto protein frameworks. PMID:24757504

  14. Site-specific Labeling of a Protein Lysine Residue By Novel Kinetic Labeling Combinatorial Libraries.

    PubMed

    Krantz, Allen; Hanel, Arthur M; Strug, Ivona; Wilczynski, Andrzej; Wolff, Jeremy J; Huang, Wolin; Huang, Linda H; Settineri, Tina; Holmes, Darren L; Hardy, Margaret C; Bridon, Dominique P

    2014-01-01

    The first example of a kinetic labeling library designed to enable the discovery of affinity labels is presented. Each library component (1) consists of a variable peptidyl component linked to a biotinyl moiety by a 4-mercaptobenzoyl linker in thioester format. We demonstrate that an affinity label can be uncovered by measuring reaction rates between library pools and the protein target, human serum albumin (HSA) and identifying significant outliers. By choosing peptide functionality compatible with a potentially reactive thioester labeling entity, libraries can be screened in pools. It is noteworthy that a limited subset of amino acids (R, S, E, F, Y, l, M, W, and Q) that compose the affinity moiety is sufficient to produce rate variances that guide the discovery process. After two rounds of deconvolution, J-FLYEE-NH2 (7-E) emerges as a bona fide affinity label of HSA. Unlike known affinity labels, the affinity moiety is not retained in the protein product, but is extruded upon acylation of the protein. This feature affords a method of introducing various payloads, without extraneous elements, onto protein frameworks. PMID:24757504

  15. Identification of a Fragment-like Small Molecule Ligand for the Methyl-lysine Binding Protein, 53BP1

    PubMed Central

    Perfetti, Michael T.; Baughma, Brandi M.; Dickson, Bradley M.; Mu, Yunxiang; Cui, Gaofeng; Mader, Pavel; Dong, Aiping; Norris, Jacqueline L.; Rothbart, Scott B.; Strahl, Brian D.; Brown, Peter J.; Janzen, William P.; Arrowsmith, Cheryl H.; Mer, Georges; McBride, Kevin M.

    2015-01-01

    Improving our understanding of the role of chromatin regulators in the initiation, development, and suppression of cancer and other devastating diseases is critical, as they are integral players in regulating DNA integrity and gene expression. Developing small molecule inhibitors for this target class with cellular activity is a crucial step toward elucidating their specific functions. We specifically targeted the DNA damage response protein, 53BP1, which uses its tandem tudor domain to recognize histone H4 dimethylated on lysine 20 (H4K20me2), a modification induced by double-strand DNA breaks. Through a cross-screening approach we identified UNC2170 (1) as a micromolar ligand of 53BP1, which demonstrates at least 17-fold selectivity for 53BP1 as compared to other methyl-lysine (Kme) binding proteins tested. Structural studies revealed that the tert-butyl amine of UNC2170 anchors the compound in the methyl-lysine (Kme) binding pocket of 53BP1, making it competitive with endogenous Kme substrates. X-ray crystallography also demonstrated that UNC2170 binds at the interface of two tudor domains of a 53BP1 dimer. Importantly, this compound functions as a 53BP1 antagonist in cellular lysates and shows cellular activity by suppressing class switch recombination, a process which requires a functional 53BP1 tudor domain. These results demonstrate that UNC2170 is a functionally active, fragment-like ligand for 53BP1. PMID:25590533

  16. Huntingtin N-Terminal Monomeric and Multimeric Structures Destabilized by Covalent Modification of Heteroatomic Residues.

    PubMed

    Arndt, James R; Kondalaji, Samaneh Ghassabi; Maurer, Megan M; Parker, Arlo; Legleiter, Justin; Valentine, Stephen J

    2015-07-21

    Early stage oligomer formation of the huntingtin protein may be driven by self-association of the 17-residue amphipathic α-helix at the protein's N-terminus (Nt17). Oligomeric structures have been implicated in neuronal toxicity and may represent important neurotoxic species in Huntington's disease. Therefore, a residue-specific structural characterization of Nt17 is crucial to understanding and potentially inhibiting oligomer formation. Native electrospray ion mobility spectrometry-mass spectrometry (IMS-MS) techniques and molecular dynamics simulations (MDS) have been applied to study coexisting monomer and multimer conformations of Nt17, independent of the remainder of huntingtin exon 1. MDS suggests gas-phase monomer ion structures comprise a helix-turn-coil configuration and a helix-extended-coil region. Elongated dimer species comprise partially helical monomers arranged in an antiparallel geometry. This stacked helical bundle may represent the earliest stages of Nt17-driven oligomer formation. Nt17 monomers and multimers have been further probed using diethylpyrocarbonate (DEPC). An N-terminal site (N-terminus of Threonine-3) and Lysine-6 are modified at higher DEPC concentrations, which led to the formation of an intermediate monomer structure. These modifications resulted in decreased extended monomer ion conformers, as well as a reduction in multimer formation. From the MDS experiments for the dimer ions, Lys6 residues in both monomer constituents interact with Ser16 and Glu12 residues on adjacent peptides; therefore, the decrease in multimer formation could result from disruption of these or similar interactions. This work provides a structurally selective model from which to study Nt17 self-association and provides critical insight toward Nt17 multimerization and, possibly, the early stages of huntingtin exon 1 aggregation. PMID:26098795

  17. Huntingtin N-terminal monomeric and multimeric structures destabilized by covalent modification of heteroatomic residues

    PubMed Central

    Arndt, James R.; Kondalaji, Samaneh G.; Maurer, Megan M.; Parker, Arlo; Legleiter, Justin

    2015-01-01

    Early-stage oligomer formation of the huntingtin protein may be driven by self-association of the seventeen-residue amphipathic α-helix at the protein’s N-terminus (Nt17). Oligomeric structures have been implicated in neuronal toxicity and may represent important neurotoxic species in Huntington’s disease. Therefore, a residue-specific structural characterization of Nt17 is crucial to understanding and potentially inhibiting oligomer formation. Native electrospray ion mobility spectrometry-mass spectrometry (IMS-MS) techniques and molecular dynamics simulations (MDS), have been applied to study coexisting monomer and multimer conformations of Nt17, independent of the remainder of huntingtin exon 1. MDS suggests gas-phase monomer ion structures are comprised of a helix-turn-coil configuration and a helix-extended coil region. Elongated dimer species are comprised of partially-helical monomers arranged in an antiparallel geometry. This stacked helical bundle may represent the earliest stages of Nt17-driven oligomer formation. Nt17 monomers and multimers have been further probed using diethylpyrocarbonate (DEPC). An N-terminal site (N-terminus of Threonine-3) and Lysine-6 are modified at higher DEPC concentrations, which led to the formation of an intermediate monomer structure. These modifications resulted in decreased extended monomer ion conformers, as well as a reduction in multimer formation. From the MDS experiments for the dimer ions, Lys6 residues in both monomer constituents interact with Ser16 and Glu12 residues on adjacent peptides; therefore, the decrease in multimer formation could result from disruption of these or similar interactions. This work provides a structurally selective model from which to study Nt17 self-association and provides critical insight toward Nt17 multimerization and possibly, the early stages of huntingtin exon 1 aggregation. PMID:26098795

  18. Copper complexes of glycyl-histidyl-lysine and two of its synthetic analogues: chemical behaviour and biological activity.

    PubMed

    Conato, C; Gavioli, R; Guerrini, R; Kozlowski, H; Mlynarz, P; Pasti, C; Pulidori, F; Remelli, M

    2001-05-01

    Copper complex formation equilibria of glycyl-L-histidyl-L-lysine (Gly-His-Lys, GHK) and of two synthetic analogues, where the histidine residue was replaced with a synthetic amino acid (L-spinacine or L-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid), have been carefully investigated using different experimental techniques: potentiometry, solution calorimetry, UV-VIS spectrophotometry, circular dichroism and electron paramagnetic resonance spectroscopies. All the ligands formed complexes having different stoichiometries and stabilities; evidence for the formation of binuclear species is also shown. The structures of the main complexes are discussed. It is suggested that the lateral lysine amino group participates in complex formation, but only at alkaline pH values: at physiological pH this group is protonated and available for possible interactions with cellular receptors. The above tripeptides have been tested for their enzymatic stability in human serum: the synthetic compounds showed no significant degradation for at least 3 h. Finally, their activity as growth factor has been studied in vitro. The two synthetic analogues showed an activity comparable to or even higher than that of GHK, thus suggesting their possible use as additives in cell culture media, even in the presence of serum. Relevant information on the GHK action mechanism as cell growth factor has been obtained: the formation of copper complexes, driven by the first (Gly) residue, appears necessary while the second residue (His) does not appear to play a specific role; the presence of the free side chain of the third residue (Lys) appears to be of fundamental importance. PMID:11325542

  19. Lysine-Based Site-Directed Mutagenesis Increased Rigid β-Sheet Structure and Thermostability of Mesophilic 1,3-1,4-β-Glucanase.

    PubMed

    Niu, Chengtuo; Zhu, Linjiang; Zhu, Pei; Li, Qi

    2015-06-01

    1,3-1,4-β-Glucanase is widely applied in the food industry, while its low thermostability often reduces its performance. In a previous study, chemical modification of surface lysine residues was proved to increase the thermostability of β-glucanase. To improve the thermostability, the mesophilic β-glucanase from Bacillus terquilensis was rationally engineered through site-directed mutagenesis of the 12 lysines into serines. The results showed that the K20S, K117S, and K165S mutants could both enhance the specific activities and thermostability of β-glucanase. The triple mutant (K20S/K117S/K165S) could increase the optimal temperature and T50 value by 15 and 14 °C, respectively. Five percent more structured residues were observed in the mutant, which formed new β-sheet structures in the concave side. Molecular dynamics simulation analysis showed that the flexibility in the mutation regions was decreased, which resulted in the overall rigidity of the β-glucanase. Therefore, the lysine-based site-directed mutagenesis is a simple and effective method for improving the thermostability of β-glucanase. PMID:25953154

  20. Sirtuin 3 (SIRT3) protein regulates long-chain acyl-CoA dehydrogenase by deacetylating conserved lysines near the active site.

    PubMed

    Bharathi, Sivakama S; Zhang, Yuxun; Mohsen, Al-Walid; Uppala, Radha; Balasubramani, Manimalha; Schreiber, Emanuel; Uechi, Guy; Beck, Megan E; Rardin, Matthew J; Vockley, Jerry; Verdin, Eric; Gibson, Bradford W; Hirschey, Matthew D; Goetzman, Eric S

    2013-11-22

    Long-chain acyl-CoA dehydrogenase (LCAD) is a key mitochondrial fatty acid oxidation enzyme. We previously demonstrated increased LCAD lysine acetylation in SIRT3 knockout mice concomitant with reduced LCAD activity and reduced fatty acid oxidation. To study the effects of acetylation on LCAD and determine sirtuin 3 (SIRT3) target sites, we chemically acetylated recombinant LCAD. Acetylation impeded substrate binding and reduced catalytic efficiency. Deacetylation with recombinant SIRT3 partially restored activity. Residues Lys-318 and Lys-322 were identified as SIRT3-targeted lysines. Arginine substitutions at Lys-318 and Lys-322 prevented the acetylation-induced activity loss. Lys-318 and Lys-322 flank residues Arg-317 and Phe-320, which are conserved among all acyl-CoA dehydrogenases and coordinate the enzyme-bound FAD cofactor in the active site. We propose that acetylation at Lys-318/Lys-322 causes a conformational change which reduces hydride transfer from substrate to FAD. Medium-chain acyl-CoA dehydrogenase and acyl-CoA dehydrogenase 9, two related enzymes with lysines at positions equivalent to Lys-318/Lys-322, were also efficiently deacetylated by SIRT3 following chemical acetylation. These results suggest that acetylation/deacetylation at Lys-318/Lys-322 is a mode of regulating fatty acid oxidation. The same mechanism may regulate other acyl-CoA dehydrogenases. PMID:24121500

  1. Sirtuin 3 (SIRT3) Protein Regulates Long-chain Acyl-CoA Dehydrogenase by Deacetylating Conserved Lysines Near the Active Site

    PubMed Central

    Bharathi, Sivakama S.; Zhang, Yuxun; Mohsen, Al-Walid; Uppala, Radha; Balasubramani, Manimalha; Schreiber, Emanuel; Uechi, Guy; Beck, Megan E.; Rardin, Matthew J.; Vockley, Jerry; Verdin, Eric; Gibson, Bradford W.; Hirschey, Matthew D.; Goetzman, Eric S.

    2013-01-01

    Long-chain acyl-CoA dehydrogenase (LCAD) is a key mitochondrial fatty acid oxidation enzyme. We previously demonstrated increased LCAD lysine acetylation in SIRT3 knockout mice concomitant with reduced LCAD activity and reduced fatty acid oxidation. To study the effects of acetylation on LCAD and determine sirtuin 3 (SIRT3) target sites, we chemically acetylated recombinant LCAD. Acetylation impeded substrate binding and reduced catalytic efficiency. Deacetylation with recombinant SIRT3 partially restored activity. Residues Lys-318 and Lys-322 were identified as SIRT3-targeted lysines. Arginine substitutions at Lys-318 and Lys-322 prevented the acetylation-induced activity loss. Lys-318 and Lys-322 flank residues Arg-317 and Phe-320, which are conserved among all acyl-CoA dehydrogenases and coordinate the enzyme-bound FAD cofactor in the active site. We propose that acetylation at Lys-318/Lys-322 causes a conformational change which reduces hydride transfer from substrate to FAD. Medium-chain acyl-CoA dehydrogenase and acyl-CoA dehydrogenase 9, two related enzymes with lysines at positions equivalent to Lys-318/Lys-322, were also efficiently deacetylated by SIRT3 following chemical acetylation. These results suggest that acetylation/deacetylation at Lys-318/Lys-322 is a mode of regulating fatty acid oxidation. The same mechanism may regulate other acyl-CoA dehydrogenases. PMID:24121500

  2. Protein architecture and core residues in unwound α-helices provide insights to the transport function of plant AtCHX17.

    PubMed

    Czerny, Daniel D; Padmanaban, Senthilkumar; Anishkin, Andriy; Venema, Kees; Riaz, Zoya; Sze, Heven

    2016-09-01

    Using Arabidopsis thaliana AtCHX17 as an example, we combine structural modeling and mutagenesis to provide insights on its protein architecture and transport function which is poorly characterized. This approach is based on the observation that protein structures are significantly more conserved in evolution than linear sequences, and mechanistic similarities among diverse transporters are emerging. Two homology models of AtCHX17 were obtained that show a protein fold similar to known structures of bacterial Na(+)/H(+) antiporters, EcNhaA and TtNapA. The distinct secondary and tertiary structure models highlighted residues at positions potentially important for CHX17 activity. Mutagenesis showed that asparagine-N200 and aspartate-D201 inside transmembrane5 (TM5), and lysine-K355 inside TM10 are critical for AtCHX17 activity. We reveal previously unrecognized threonine-T170 and lysine-K383 as key residues at unwound regions in the middle of TM4 and TM11 α-helices, respectively. Mutation of glutamate-E111 located near the membrane surface inhibited AtCHX17 activity, suggesting a role in pH sensing. The long carboxylic tail of unknown purpose has an alternating β-sheet and α-helix secondary structure that is conserved in prokaryote universal stress proteins. These results support the overall architecture of AtCHX17 and identify D201, N200 and novel residues T170 and K383 at the functional core which likely participates in ion recognition, coordination and/or translocation, similar to characterized cation/H(+) exchangers. The core of AtCHX17 models according to EcNhaA and TtNapA templates faces inward and outward, respectively, which may reflect two conformational states of the alternating access transport mode for proteins belonging to the plant CHX family. PMID:27179641

  3. Structure and Histone Binding Properties of the Vps75-Rtt109 Chaperone-Lysine Acetyltransferase Complex

    SciTech Connect

    Su, Dan; Hu, Qi; Zhou, Hui; Thompson, James R.; Xu, Rui-Ming; Zhang, Zhiguo; Mer, Georges

    2011-11-02

    The histone chaperone Vps75 presents the remarkable property of stimulating the Rtt109-dependent acetylation of several histone H3 lysine residues within (H3-H4){sub 2} tetramers. To investigate this activation mechanism, we determined x-ray structures of full-length Vps75 in complex with full-length Rtt109 in two crystal forms. Both structures show similar asymmetric assemblies of a Vps75 dimer bound to an Rtt109 monomer. In the Vps75-Rtt109 complexes, the catalytic site of Rtt109 is confined to an enclosed space that can accommodate the N-terminal tail of histone H3 in (H3-H4){sub 2}. Investigation of Vps75-Rtt109-(H3-H4)2 and Vps75-(H3-H4)2 complexes by NMR spectroscopy-probed hydrogen/deuterium exchange suggests that Vps75 guides histone H3 in the catalytic enclosure. These findings clarify the basis for the enhanced acetylation of histone H3 tail residues by Vps75-Rtt109.

  4. Structural insights into the mechanism of four-coordinate Cob(II)alamin formation in the active site of the Salmonella enterica ATP:Co(I)rrinoid adenosyltransferase enzyme: critical role of residues Phe91 and Trp93.

    PubMed

    Moore, Theodore C; Newmister, Sean A; Rayment, Ivan; Escalante-Semerena, Jorge C

    2012-12-01

    ATP:co(I)rrinoid adenosyltransferases (ACATs) are enzymes that catalyze the formation of adenosylcobalamin (AdoCbl, coenzyme B(12)) from cobalamin and ATP. There are three families of ACATs, namely, CobA, EutT, and PduO. In Salmonella enterica, CobA is the housekeeping enzyme that is required for de novo AdoCbl synthesis and for salvaging incomplete precursors and cobalamin from the environment. Here, we report the crystal structure of CobA in complex with ATP, four-coordinate cobalamin, and five-coordinate cobalamin. This provides the first crystallographic evidence of the existence of cob(II)alamin in the active site of CobA. The structure suggests a mechanism in which the enzyme adopts a closed conformation and two residues, Phe91 and Trp93, displace 5,6-dimethylbenzimidazole, the lower nucleotide ligand base of cobalamin, to generate a transient four-coordinate cobalamin, which is critical in the formation of the AdoCbl Co-C bond. In vivo and in vitro mutational analyses of Phe91 and Trp93 emphasize the important role of bulky hydrophobic side chains in the active site. The proposed manner in which CobA increases the redox potential of the cob(II)alamin/cob(I)alamin couple to facilitate formation of the Co-C bond appears to be analogous to that utilized by the PduO-type ACATs, where in both cases the polar coordination of the lower ligand to the cobalt ion is eliminated by placing that face of the corrin ring adjacent to a cluster of bulky hydrophobic side chains. PMID:23148601

  5. Characterization of a Linked Jumonji Domain of the KDM5/JARID1 Family of Histone H3 Lysine 4 Demethylases.

    PubMed

    Horton, John R; Engstrom, Amanda; Zoeller, Elizabeth L; Liu, Xu; Shanks, John R; Zhang, Xing; Johns, Margaret A; Vertino, Paula M; Fu, Haian; Cheng, Xiaodong

    2016-02-01

    The KDM5/JARID1 family of Fe(II)- and α-ketoglutarate-dependent demethylases remove methyl groups from tri- and dimethylated lysine 4 of histone H3. Accumulating evidence from primary tumors and model systems supports a role for KDM5A (JARID1A/RBP2) and KDM5B (JARID1B/PLU1) as oncogenic drivers. The KDM5 family is unique among the Jumonji domain-containing histone demethylases in that there is an atypical insertion of a DNA-binding ARID domain and a histone-binding PHD domain into the Jumonji domain, which separates the catalytic domain into two fragments (JmjN and JmjC). Here we demonstrate that internal deletion of the ARID and PHD1 domains has a negligible effect on in vitro enzymatic kinetics of the KDM5 family of enzymes. We present a crystal structure of the linked JmjN-JmjC domain from KDM5A, which reveals that the linked domain fully reconstitutes the cofactor (metal ion and α-ketoglutarate) binding characteristics of other structurally characterized Jumonji domain demethylases. Docking studies with GSK-J1, a selective inhibitor of the KDM6/KDM5 subfamilies, identify critical residues for binding of the inhibitor to the reconstituted KDM5 Jumonji domain. Further, we found that GSK-J1 inhibited the demethylase activity of KDM5C with 8.5-fold increased potency compared with that of KDM5B at 1 mm α-ketoglutarate. In contrast, JIB-04 (a pan-inhibitor of the Jumonji demethylase superfamily) had the opposite effect and was ~8-fold more potent against KDM5B than against KDM5C. Interestingly, the relative selectivity of JIB-04 toward KDM5B over KDM5C in vitro translates to a ~10-50-fold greater growth-inhibitory activity against breast cancer cell lines. These data define the minimal requirements for enzymatic activity of the KDM5 family to be the linked JmjN-JmjC domain coupled with the immediate C-terminal helical zinc-binding domain and provide structural characterization of the linked JmjN-JmjC domain for the KDM5 family, which should prove useful in the

  6. Progress in the Development of Lysine Methyltransferase SETD8 Inhibitors.

    PubMed

    Milite, Ciro; Feoli, Alessandra; Viviano, Monica; Rescigno, Donatella; Mai, Antonello; Castellano, Sabrina; Sbardella, Gianluca

    2016-08-19

    SETD8/SET8/Pr-SET7/KMT5A is the only known lysine methyltransferase that monomethylates lysine 20 of histone H4 (H4K20) in vivo. The methyltransferase activity of SETD8 has been implicated in many essential cellular processes, including DNA replication, DNA damage response, transcription modulation, and cell cycle regulation. In addition to H4K20, SETD8 monomethylates non-histone substrates including proliferating cell nuclear antigen and p53. During the past decade, different structural classes of inhibitors targeting various lysine methyltransferases have been designed and developed. However, the development of SETD8 inhibitors is still in its infancy. This review covers the progress made to date in inhibiting the activity of SETD8 by small molecules, with an emphasis on their discovery, selectivity over other methyltransferases, and cellular activity. PMID:27411844

  7. Coacervate-like microspheres from lysine-rich proteinoid

    NASA Technical Reports Server (NTRS)

    Rohlfing, D. L.

    1975-01-01

    Microspheres form isothermally from lysine-rich proteinoid when the ionic strength of the solution is increased with NaCl or other salts. Studies with different monovalent anions and with polymers of different amino acid composition indicate that charge neutralization and hydrophobic bonding contribute to microsphere formation. The particles also form in sea water, especially if heated or made slightly alkaline. The microspheres differ from those made from acidic proteinoid but resemble coacervate droplets in some ways (isothermal formation, limited stability, stabilization by quinone, uptake of dyes). Because the constituent lysine-rich proteinoid is of simulated prebiotic origin, the study is interpreted to add emphasis to and suggest an evolutionary continuity for coacervation phenomena.

  8. The self-assembly of a camptothecin-lysine nanotube.

    PubMed

    Sun, Yuan; Shieh, Aileen; Kim, Se Hye; King, Samantha; Kim, Anne; Sun, Hui-Lung; Croce, Carlo M; Parquette, Jon R

    2016-06-15

    A simple, low molecular weight camptothecin-lysine conjugate is reported to self-assemble into nanotubes with diameters of 70-100nm and a drug loading level of 60.5%. The nanotubes exhibited promising in vitro cytotoxicity against cancer cell lines A549, NCI-H460 and NCI-H23. The release of active camptothecin was highly dependent on conjugate concentration, temperature and pH of the solution. PMID:27156772

  9. Discovery and Characterization of a Highly Potent and Selective Aminopyrazoline-Based in Vivo Probe (BAY-598) for the Protein Lysine Methyltransferase SMYD2

    PubMed Central

    2016-01-01

    Protein lysine methyltransferases have recently emerged as a new target class for the development of inhibitors that modulate gene transcription or signaling pathways. SET and MYND domain containing protein 2 (SMYD2) is a catalytic SET domain containing methyltransferase reported to monomethylate lysine residues on histone and nonhistone proteins. Although several studies have uncovered an important role of SMYD2 in promoting cancer by protein methylation, the biology of SMYD2 is far from being fully understood. Utilization of highly potent and selective chemical probes for target validation has emerged as a concept which circumvents possible limitations of knockdown experiments and, in particular, could result in an improved exploration of drug targets with a complex underlying biology. Here, we report the development of a potent, selective, and cell-active, substrate-competitive inhibitor of SMYD2, which is the first reported inhibitor suitable for in vivo target validation studies in rodents. PMID:27075367

  10. Discovery and Characterization of a Highly Potent and Selective Aminopyrazoline-Based in Vivo Probe (BAY-598) for the Protein Lysine Methyltransferase SMYD2.

    PubMed

    Eggert, Erik; Hillig, Roman C; Koehr, Silke; Stöckigt, Detlef; Weiske, Jörg; Barak, Naomi; Mowat, Jeffrey; Brumby, Thomas; Christ, Clara D; Ter Laak, Antonius; Lang, Tina; Fernandez-Montalvan, Amaury E; Badock, Volker; Weinmann, Hilmar; Hartung, Ingo V; Barsyte-Lovejoy, Dalia; Szewczyk, Magdalena; Kennedy, Steven; Li, Fengling; Vedadi, Masoud; Brown, Peter J; Santhakumar, Vijayaratnam; Arrowsmith, Cheryl H; Stellfeld, Timo; Stresemann, Carlo

    2016-05-26

    Protein lysine methyltransferases have recently emerged as a new target class for the development of inhibitors that modulate gene transcription or signaling pathways. SET and MYND domain containing protein 2 (SMYD2) is a catalytic SET domain containing methyltransferase reported to monomethylate lysine residues on histone and nonhistone proteins. Although several studies have uncovered an important role of SMYD2 in promoting cancer by protein methylation, the biology of SMYD2 is far from being fully understood. Utilization of highly potent and selective chemical probes for target validation has emerged as a concept which circumvents possible limitations of knockdown experiments and, in particular, could result in an improved exploration of drug targets with a complex underlying biology. Here, we report the development of a potent, selective, and cell-active, substrate-competitive inhibitor of SMYD2, which is the first reported inhibitor suitable for in vivo target validation studies in rodents. PMID:27075367

  11. Phytate utilization by genetically engineered lysine-producing Corynebacterium glutamicum.

    PubMed

    Tzvetkov, Mladen V; Liebl, Wolfgang

    2008-04-30

    Heterologous expression of a phytase gene (phyC) from Bacillus amyloliquefaciens DSM 7 enabled the growth of Corynebacterium glutamicum with phytate (myo-inositol-1,2,3,4,5,6-hexakisphosphate) as a new, sole source of phosphorus. Phytate was not used as a carbon source. During growth of the phyC-expressing amino acid (l-lysine)-producing strain C. glutamicum ATCC 21253 (pWLQ2::phyC) with phytate as the source of phosphorus, merely a small, transient accumulation of inorganic phosphate was observed in the fermentation broth. At the later stages of fermentation, free inorganic phosphate from phytate degradation was no longer detectable. Growth and l-lysine production by the phytase-producing strain C. glutamicum ATCC 21253 (pWLQ2::phyC) in phytate medium did not differ significantly from control experiments with strain C. glutamicum ATCC 21253 (pWLQ2) conducted with an excess of inorganic phosphate, demonstrating that there was no phosphate limitation when phytate was used as the phosphorus source. Under the expression conditions employed, only part of PhyC was secreted to the culture broth by C. glutamicum, but this did not significantly affect growth or lysine production. PMID:18374441

  12. Proteome-wide enrichment of proteins modified by lysine methylation

    PubMed Central

    Carlson, Scott M; Moore, Kaitlyn E; Green, Erin M; Martín, Glòria Mas; Gozani, Or

    2015-01-01

    We present a protocol for using the triple malignant brain tumor domains of L3MBTL1 (3×MBT), which bind to mono- and di-methylated lysine with minimal sequence specificity, in order to enrich for such methylated lysine from cell lysates. Cells in culture are grown with amino acids containing light or heavy stable isotopic labels. Methylated proteins are enriched by incubating cell lysates with 3×MBT, or with the binding-null D355N mutant as a negative control. Quantitative liquid chromatography and tandem mass spectrometry (LC-MS/MS) are then used to identify proteins that are specifically enriched by 3×MBT pull-down. The addition of a third isotopic label allows the comparison of protein lysine methylation between different biological conditions. Unlike most approaches, our strategy does not require a prior hypothesis of candidate methylated proteins, and it recognizes a wider range of methylated proteins than any available method using antibodies. Cells are prepared by growing in isotopic labeling medium for about 7 d; the process of enriching methylated proteins takes 3 d and analysis by LC-MS/MS takes another 1–2 d. PMID:24309976

  13. Expression of the Escherichia coli pntAB genes encoding a membrane-bound transhydrogenase in Corynebacterium glutamicum improves L-lysine formation.

    PubMed

    Kabus, Armin; Georgi, Tobias; Wendisch, Volker F; Bott, Michael

    2007-05-01

    A critical factor in the biotechnological production of L: -lysine with Corynebacterium glutamicum is the sufficient supply of NADPH. The membrane-integral nicotinamide nucleotide transhydrogenase PntAB of Escherichia coli can use the electrochemical proton gradient across the cytoplasmic membrane to drive the reduction of NADP(+) via the oxidation of NADH. As C. glutamicum does not possess such an enzyme, we expressed the E. coli pntAB genes in the genetically defined C. glutamicum lysine-producing strain DM1730, resulting in membrane-associated transhydrogenase activity of 0.7 U/mg protein. When cultivated in minimal medium with 10% (w/v) carbon source, the presence of transhydrogenase slightly reduced glucose consumption, whereas the consumption of fructose, glucose plus fructose, and, in particular, sucrose was stimulated. Biomass was increased by pntAB expression between 10 and 30% on all carbon sources tested. Most importantly, the lysine concentration was increased in the presence of transhydrogenase by approximately 10% on glucose, approximately 70% on fructose, approximately 50% on glucose plus fructose, and even by approximately 300% on sucrose. Thus, the presence of a proton-coupled transhydrogenase was shown to be an efficient way to improve lysine production by C. glutamicum. In contrast, pntAB expression had a negative effect on growth and glutamate production of C. glutamicum wild type. PMID:17216441

  14. The Evolutionarily Conserved Tre2/Bub2/Cdc16 (TBC), Lysin Motif (LysM), Domain Catalytic (TLDc) Domain Is Neuroprotective against Oxidative Stress*

    PubMed Central

    Finelli, Mattéa J.; Sanchez-Pulido, Luis; Liu, Kevin X; Davies, Kay E.; Oliver, Peter L.

    2016-01-01

    Oxidative stress is a pathological feature of many neurological disorders; therefore, utilizing proteins that are protective against such cellular insults is a potentially valuable therapeutic approach. Oxidation resistance 1 (OXR1) has been shown previously to be critical for oxidative stress resistance in neuronal cells; deletion of this gene causes neurodegeneration in mice, yet conversely, overexpression of OXR1 is protective in cellular and mouse models of amyotrophic lateral sclerosis. However, the molecular mechanisms involved are unclear. OXR1 contains the Tre2/Bub2/Cdc16 (TBC), lysin motif (LysM), domain catalytic (TLDc) domain, a motif present in a family of proteins including TBC1 domain family member 24 (TBC1D24), a protein mutated in a range of disorders characterized by seizures, hearing loss, and neurodegeneration. The TLDc domain is highly conserved across species, although the structure-function relationship is unknown. To understand the role of this domain in the stress response, we carried out systematic analysis of all mammalian TLDc domain-containing proteins, investigating their expression and neuroprotective properties in parallel. In addition, we performed a detailed structural and functional study of this domain in which we identified key residues required for its activity. Finally, we present a new mouse insertional mutant of Oxr1, confirming that specific disruption of the TLDc domain in vivo is sufficient to cause neurodegeneration. Our data demonstrate that the integrity of the TLDc domain is essential for conferring neuroprotection, an important step in understanding the functional significance of all TLDc domain-containing proteins in the cellular stress response and disease. PMID:26668325

  15. The Different Inhibition Mechanisms of OXA-1 and OXA-24 β-Lactamases Are Determined by the Stability of Active Site Carboxylated Lysine*

    PubMed Central

    Che, Tao; Bethel, Christopher R.; Pusztai-Carey, Marianne; Bonomo, Robert A.; Carey, Paul R.

    2014-01-01

    The catalytic efficiency of class D β-lactamases depends critically on an unusual carboxylated lysine as the general base residue for both the acylation and deacylation steps of the enzyme. Microbiological and biochemical studies on the class D β-lactamases OXA-1 and OXA-24 showed that the two enzymes behave differently when reacting with two 6-methylidene penems (penem 1 and penem 3): the penems are good inhibitors of OXA-1 but act more like substrates for OXA-24. UV difference and Raman spectroscopy revealed that the respective reaction mechanisms are different. The penems form an unusual intermediate, a 1,4-thiazepine derivative in OXA-1, and undergo deacylation followed by the decarboxylation of Lys-70, rendering OXA-1 inactive. This inactivation could not be reversed by the addition of 100 mm NaHCO3. In OXA-24, under mild conditions (enzyme:inhibitor = 1:4), only hydrolyzed products were detected, and the enzyme remained active. However, under harsh conditions (enzyme:inhibitor = 1:2000), OXA-24 was inhibited via decarboxylation of Lys-84; however, the enzyme could be reactivated by the addition of 100 mm NaHCO3. We conclude that OXA-24 not only decarboxylates with difficulty but also recarboxylates with ease; in contrast, OXA-1 decarboxylates easily but recarboxylates with difficulty. Structural analysis of the active site indicates that a crystallographic water molecule may play an important role in carboxylation in OXA-24 (an analogous water molecule is not found in OXA-1), supporting the suggestion that a water molecule in the active site of OXA-24 can lower the energy barrier for carboxylation significantly. PMID:24443569

  16. Residual Cap

    NASA Technical Reports Server (NTRS)

    2006-01-01

    10 May 2006 This Mars Global Surveyor (MGS) Mars Orbiter Camera (MOC) image shows a summertime view of the south polar residual cap of Mars. In this image, mesas composed largely of solid carbon dioxide are separated from one another by irregularly-shaped depressions. The variation in brightness across this scene is a function of several factors including, but not limited to, varying proportions of dust and solid carbon dioxide, undulating topography, and differences in the roughness of the slopes versus the flat surfaces.

    Location near: 86.7oS, 343.3oW Image width: 3 km (1.9 mi) Illumination from: upper left Season: Southern Summer

  17. Mammalian Protein Arginine Methyltransferase 7 (PRMT7) Specifically Targets RXR Sites in Lysine- and Arginine-rich Regions*

    PubMed Central

    Feng, You; Maity, Ranjan; Whitelegge, Julian P.; Hadjikyriacou, Andrea; Li, Ziwei; Zurita-Lopez, Cecilia; Al-Hadid, Qais; Clark, Amander T.; Bedford, Mark T.; Masson, Jean-Yves; Clarke, Steven G.

    2013-01-01

    The mammalian protein arginine methyltransferase 7 (PRMT7) has been implicated in roles of transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation, and metastasis. However, the type of reaction that it catalyzes and its substrate specificity remain controversial. In this study, we purified a recombinant mouse PRMT7 expressed in insect cells that demonstrates a robust methyltransferase activity. Using a variety of substrates, we demonstrate that the enzyme only catalyzes the formation of ω-monomethylarginine residues, and we confirm its activity as the prototype type III protein arginine methyltransferase. This enzyme is active on all recombinant human core histones, but histone H2B is a highly preferred substrate. Analysis of the specific methylation sites within intact histone H2B and within H2B and H4 peptides revealed novel post-translational modification sites and a unique specificity of PRMT7 for methylating arginine residues in lysine- and arginine-rich regions. We demonstrate that a prominent substrate recognition motif consists of a pair of arginine residues separated by one residue (RXR motif). These findings will significantly accelerate substrate profile analysis, biological function study, and inhibitor discovery for PRMT7. PMID:24247247

  18. Complete genome sequence of Corynebacterium glutamicum B253, a Chinese lysine-producing strain.

    PubMed

    Wu, Yong; Li, Pengpeng; Zheng, Ping; Zhou, Wenjuan; Chen, Ning; Sun, Jibin

    2015-08-10

    We disclosed the complete genome sequence of Corynebacterium glutamicum B253, an important lysine-producing strain in China. The genome consists a circular chromosome (3,159,203bp) and a plasmid (24,775bp), encoding 2767 protein coding genes in total. The genome contains all genes for lysine biosynthesis, and some mutations potentially relevant to lysine production were detected in comparison with sequence of other C. glutamicum. PMID:25953304

  19. Histone lysine methyltransferase Ezh1 promotes TLR-triggered inflammatory cytokine production by suppressing Tollip.

    PubMed

    Liu, Yiqi; Zhang, Qian; Ding, Yuanyuan; Li, Xia; Zhao, Dezhi; Zhao, Kai; Guo, Zhenhong; Cao, Xuetao

    2015-03-15

    Histone modifications play critical roles in the regulation of gene expression; however, their roles in the regulation of the innate response remain to be fully investigated. Using transcriptome analysis of mouse immature dendritic cells (DCs) and LPS-induced mature DCs, we identified that Ezh1 was the most upregulated histone methyltransferase during DC maturation. In this study, we investigated the role of Ezh1 in regulating the innate immune response. We found that silencing of Ezh1 significantly suppressed TLR-triggered production of cytokines, including IL-6, TNF-α, and IFN-β, in DCs and macrophages. Accordingly, TLR-activated signaling pathways were impaired in Ezh1-silenced macrophages. By transcriptome analysis of Ezh1-silenced macrophages, we found that Toll-interacting protein (Tollip), one well-known negative regulator of TLR signaling, was upregulated. Silencing of Tollip rescued TLR-triggered cytokine production in Ezh1-silenced macrophages. The SET domain of Ezh1 is essential for its enhancing effect on the TLR-triggered innate immune response and downstream signaling, indicating that Ezh1 promotes a TLR-triggered innate response through its lysine methyltransferase activity. Finally, Ezh1 was found to suppress the transcription of Tollip by directly targeting the proximal promoter of tollip and maintaining the high level of trimethylation of histone H3 lysine 27 there. Therefore, Ezh1 promotes TLR-triggered inflammatory cytokine production by suppressing the TLR negative regulator Tollip, contributing to full activation of the innate immune response against invading pathogens. PMID:25687760

  20. A few positively charged residues slow movement of a polypeptide chain across the endoplasmic reticulum membrane.

    PubMed

    Yamagishi, Marifu; Onishi, Yukiko; Yoshimura, Shotaro; Fujita, Hidenobu; Imai, Kenta; Kida, Yuichiro; Sakaguchi, Masao

    2014-08-26

    Many polypeptide chains are translocated across and integrated into the endoplasmic reticulum membrane through protein-conducting channels. During the process, amino acid sequences of translocating polypeptide chains are scanned by the channels and classified to be retained in the membrane or translocated into the lumen. We established an experimental system with which the kinetic effect of each amino acid residue on the polypeptide chain movement can be analyzed with a time resolution of tens of seconds. Positive charges greatly slow movement; only two lysine residues caused a remarkable slow down, and their effects were additive. The lysine residue was more effective than arginine. In contrast, clusters comprising three residues of each of the other 18 amino acids had little effect on chain movement. We also demonstrated that a four lysine cluster can exert the effect after being fully exposed from the ribosome. We concluded that as few as two to three residues of positively charged amino acids can slow the movement of the nascent polypeptide chain across the endoplasmic reticulum membrane. This effect provides a fundamental basis of the topogenic function of positively charged amino acids. PMID:25093244

  1. The abalone egg vitelline envelope receptor for sperm lysin is a giant multivalent molecule

    PubMed Central

    Swanson, Willie J.; Vacquier, Victor D.

    1997-01-01

    Abalone sperm lysin is a 16-kDa acrosomal protein, which nonenzymatically and species selectively creates a hole in the egg vitelline envelope (VE) through which the sperm passes to reach the egg cell membrane. The crystal structures of both monomeric and dimeric lysins have been solved and the sequences of lysins from 20 abalone species have been determined. As a first step in understanding the molecular mechanism by which lysin creates a hole in the VE, its VE receptor was isolated. The VE receptor for lysin (VERL) is an unbranched, rod-like molecule with an approximate relative molecular mass of 2 million; half the mass being carbohydrate. Fluorescence polarization studies showed positive cooperativity in the binding of lysin to VERL (EC50 ≈9 nM) and were consistent with the species selectivity of lysin in dissolving VEs. Each molecule of VERL bound between 126 and 142 molecules of monomeric lysin (two independent assays), showing that VERL possesses repetitive lysin-binding motifs. PMID:9192632

  2. Growth and antioxidant status of broilers fed supplemental lysine and pyridoxine under high ambient temperature

    PubMed Central

    Khakpour Irani, Farzaneh; Daneshyar, Mohsen; Najafi, Ramin

    2015-01-01

    Three levels of lysine (90, 100 and 110% of Ross requirement) and of pyridoxine (3, 6 and 9 mg kg-1) were used in a 3 × 3 factorial experiment to investigate the growth and blood antioxidant ability of broilers under high ambient temperature. None of the dietary supplements affected the weight gain during the starter and grower periods. Although no significant differences were detected between the treatments during the entire period, high lysine level fed birds had a lower weight gain. At any levels of pyridoxine, high lysine fed birds were lighter than others. Neither the lysine nor pyridoxine changed the feed intake or feed conversion ratio during the starter, grower and entire period. However there was no significant difference between the treatments for blood malondialdehyde (MDA) concentration, medium lysine fed birds had lower blood MDA than other ones. No significant effects on blood triglyceride, total protein and blood superoxide dismutase activity were indicated with addition of any lysine or pyridoxine level. Medium lysine fed birds had decreased blood glutathione peroxidase activity compared to the birds of other treatments. It was concluded that providing the proposed dietary lysine requirement of Ross strain during heat stress ensuring the best body weight gain and body antioxidant ability. Higher lysine level causes the retarded weight gain due to higher excretion of arginine from the body and consequently higher lipid peroxidation. PMID:26261713

  3. Inhibition of the acetyl lysine-binding pocket of bromodomain and extraterminal domain proteins interferes with adipogenesis.

    PubMed

    Goupille, Olivier; Penglong, Tipparat; Kadri, Zahra; Granger-Locatelli, Marine; Fucharoen, Suthat; Maouche-Chrétien, Leila; Prost, Stéphane; Leboulch, Philippe; Chrétien, Stany

    2016-04-15

    The bromodomain and extraterminal (BET) domain family proteins are epigenetic modulators involved in the reading of acetylated lysine residues. The first BET protein inhibitor to be identified, (+)-JQ1, a thienotriazolo-1, 4-diazapine, binds selectively to the acetyl lysine-binding pocket of BET proteins. We evaluated the impact on adipogenesis of this druggable targeting of chromatin epigenetic readers, by investigating the physiological consequences of epigenetic modifications through targeting proteins binding to chromatin. JQ1 significantly inhibited the differentiation of 3T3-L1 preadipocytes into white and brown adipocytes by down-regulating the expression of genes involved in adipogenesis, particularly those encoding the peroxisome proliferator-activated receptor (PPAR-γ), the CCAAT/enhancer-binding protein (C/EBPα) and, STAT5A and B. The expression of a constitutively activated STAT5B mutant did not prevent inhibition by JQ1. Thus, the association of BET/STAT5 is required for adipogenesis but STAT5 transcription activity is not the only target of JQ1. Treatment with JQ1 did not lead to the conversion of white adipose tissue into brown adipose tissue (BAT). BET protein inhibition thus interferes with generation of adipose tissue from progenitors, confirming the importance of the connections between epigenetic mechanisms and specific adipogenic transcription factors. PMID:26972250

  4. The Acetyl Group Buffering Action of Carnitine Acetyltransferase Offsets Macronutrient-induced Lysine Acetylation of Mitochondrial Proteins

    PubMed Central

    Davies, Michael N.; Kjalarsdottir, Lilja; Thompson, J. Will; Dubois, Laura G.; Stevens, Robert D.; Ilkayeva, Olga R.; Brosnan, M. Julia; Rolph, Timothy P.; Grimsrud, Paul A.; Muoio, Deborah M.

    2016-01-01

    Lysine acetylation (AcK), a posttranslational modification wherein a two-carbon acetyl group binds covalently to a lysine residue, occurs prominently on mitochondrial proteins and has been linked to metabolic dysfunction. An emergent theory suggests mitochondrial AcK occurs via mass action rather than targeted catalysis. To test this hypothesis we performed mass spectrometry-based acetylproteomic analyses of quadriceps muscles from mice with skeletal muscle-specific deficiency of carnitine acetyltransferase (CrAT), an enzyme that buffers the mitochondrial acetyl-CoA pool by converting short-chain acyl-CoAs to their membrane permeant acylcarnitine counterparts. CrAT deficiency increased tissue acetyl-CoA levels and susceptibility to diet-induced AcK of broad-ranging mitochondrial proteins, coincident with diminished whole body glucose control. Sub-compartment acetylproteome analyses of muscles from obese mice and humans showed remarkable overrepresentation of mitochondrial matrix proteins. These findings reveal roles for CrAT and L-carnitine in modulating the muscle acetylproteome and provide strong experimental evidence favoring the nonenzymatic carbon pressure model of mitochondrial AcK. PMID:26748706

  5. Structural basis of SETD6-mediated regulation of the NF-kB network via methyl-lysine signaling

    PubMed Central

    Chang, Yanqi; Levy, Dan; Horton, John R.; Peng, Junmin; Zhang, Xing; Gozani, Or; Cheng, Xiaodong

    2011-01-01

    SET domain containing 6 (SETD6) monomethylates the RelA subunit of nuclear factor kappa B (NF-κB). The ankyrin repeats of G9a-like protein (GLP) recognizes RelA monomethylated at Lys310. Adjacent to Lys310 is Ser311, a known phosphorylation site of RelA. Ser311 phosphorylation inhibits Lys310 methylation by SETD6 as well as binding of Lys310me1 by GLP. The structure of SETD6 in complex with RelA peptide containing the methylation site, in the presence of S-adenosyl-l-methionine, reveals a V-like protein structure and suggests a model for NF-κB binding to SETD6. In addition, structural modeling of the GLP ankyrin repeats bound to Lys310me1 peptide provides insight into the molecular basis for inhibition of Lys310me1 binding by Ser311 phosphorylation. Together, these findings provide a structural explanation for a key cellular signaling pathway centered on RelA Lys310 methylation, which is generated by SETD6 and recognized by GLP, and incorporate a methylation–phosphorylation switch of adjacent lysine and serine residues. Finally, SETD6 is structurally similar to the Rubisco large subunit methyltransferase. Given the restriction of Rubisco to plant species, this particular appearance of the protein lysine methyltransferase has been evolutionarily well conserved. PMID:21515635

  6. Acetylation of Lysine 201 Inhibits the DNA-Binding Ability of PhoP to Regulate Salmonella Virulence.

    PubMed

    Ren, Jie; Sang, Yu; Tan, Yongcong; Tao, Jing; Ni, Jinjing; Liu, Shuting; Fan, Xia; Zhao, Wei; Lu, Jie; Wu, Wenjuan; Yao, Yu-Feng

    2016-03-01

    The two-component system PhoP-PhoQ is highly conserved in bacteria and regulates virulence in response to various signals for bacteria within the mammalian host. Here, we demonstrate that PhoP could be acetylated by Pat and deacetylated by deacetylase CobB enzymatically in vitro and in vivo in Salmonella Typhimurium. Specifically, the conserved lysine residue 201(K201) in winged helix-turn-helix motif at C-terminal DNA-binding domain of PhoP could be acetylated, and its acetylation level decreases dramatically when bacteria encounter low magnesium, acid stress or phagocytosis of macrophages. PhoP has a decreased acetylation and increased DNA-binding ability in the deletion mutant of pat. However, acetylation of K201 does not counteract PhoP phosphorylation, which is essential for PhoP activity. In addition, acetylation of K201 (mimicked by glutamine substitute) in S. Typhimurium causes significantly attenuated intestinal inflammation as well as systemic infection in mouse model, suggesting that deacetylation of PhoP K201 is essential for Salmonella pathogenesis. Therefore, we propose that the reversible acetylation of PhoP K201 may ensure Salmonella promptly respond to different stresses in host cells. These findings suggest that reversible lysine acetylation in the DNA-binding domain, as a novel regulatory mechanism of gene expression, is involved in bacterial virulence across microorganisms. PMID:26943369

  7. Existence of Separate Domains in Lysin PlyG for Recognizing Bacillus anthracis Spores and Vegetative Cells

    PubMed Central

    Yang, Hang; Wang, Dian-Bing; Dong, Qiuhua; Zhang, Zhiping; Cui, Zongqiang; Deng, Jiaoyu; Yu, Junping

    2012-01-01

    As a potential antimicrobial, the bacteriophage lysin PlyG has been reported to specifically recognize Bacillus anthracis vegetative cells only and to kill B. anthracis vegetative cells and its germinating spores. However, how PlyG interacts with B. anthracis spores remains unclear. Herein, a 60-amino-acid domain in PlyG (residues 106 to 165), located mainly in the previously identified catalytic domain, was found able to specifically recognize B. anthracis spores but not vegetative cells. The exosporium of the spores was found to be the most probable binding target of this domain. This is the first time that a lysin for spore-forming bacteria has been found to have separate domains to recognize spores and vegetative cells, which might help in understanding the coevolution of phages with spore-forming bacteria. Besides providing new biomarkers for developing better assays for identifying B. anthracis spores, the newly found domain may be helpful in developing PlyG as a preventive antibiotic to reduce the threat of anthrax in suspected exposures to B. anthracis spores. PMID:22802245

  8. Solid-Phase Synthesis with Attachment of Peptide to Resin through an Amino Acid Side Chain: [8-Lysine]-Vasopressin

    PubMed Central

    Meienhofer, Johannes; Trzeciak, Arnold

    1971-01-01

    It is proposed that the scope of solid-phase peptide synthesis could be considerably broadened by attaching peptides to the solid-phase through functional side-chain groups rather than through the commonly used α-carboxyl groups. Side-chain attachment offers the use of a large variety of chemical linkages to solid supports. Attachment through the ε-amino group of the lysine residue to a polystyrene resin has been applied to a solid-phase synthesis of lysine-vasopressin. Nα-tert-butyl-oxycarbonyl-L-lysyl-glycinamide was condensed with chloroformoxymethyl polystyrene-2% divinylbenzene resin. After removal of the Nα-protecting tert-butyloxycarbonyl group, the peptide chain was elongated by standard Merrifield procedures to give Tos-Cys(Bzl)-Tyr-Phe-Glu-(NH2) - Asp(NH2) - Cys(Bzl) - Pro - Lys(Z - resin) - Gly-NH2. Cleavage from the resin with HBr in dioxane or trifluoroacetic acid gave a partially protected nonapeptide hydrobromide. For purification, it was converted into a fully protected peptide by treatment with benzyl p-nitro-phenyl carbonate and crystallized. Deprotection by sodium in liquid ammonia, oxidative cyclization, IRC-50 desalting, and ion-exchange chromatography gave lysinevasopressin with high potency in a rat-pressor assay. PMID:5280519

  9. Peptide-induced membrane leakage by lysine derivatives of gramicidin A in liposomes, planar bilayers, and erythrocytes.

    PubMed

    Sorochkina, Alexandra I; Kovalchuk, Sergei I; Omarova, Elena O; Sobko, Alexander A; Kotova, Elena A; Antonenko, Yuri N

    2013-11-01

    Introducing a charged group near the N-terminus of gramicidin A (gA) is supposed to suppress its ability to form ion channels by restricting its head-to-head dimerization. The present study dealt with the activity of [Lys1]gA, [Lys3]gA, [Glu1]gA, [Glu3]gA, [Lys2]gA, and [Lys5]gA in model membrane systems (planar lipid bilayers and liposomes) and erythrocytes. In contrast to the Glu-substituted peptides, the lysine derivatives of gA caused non-specific liposomal leakage monitored by fluorescence dequenching of lipid vesicles loaded with carboxyfluorescein or other fluorescent dyes. Measurements of electrical current through a planar lipid membrane revealed formation of giant pores by Lys-substituted analogs, which depended on the presence of solvent in the bilayer lipid membrane. The efficacy of unselective pore formation in liposomes depended on the position of the lysine residue in the amino acid sequence, increasing in the row: [Lys2]gA<[Lys5]gA<[Lys1]gA<[Lys3]gA. The similar series of potency was exhibited by the Lys-substituted gA analogs in facilitating erythrocyte hemolysis, whereas the Glu-substituted analogs showed negligible hemolytic activity. Oligomerization of the Lys-substituted peptides is suggested to be involved in the process of nonselective pore formation. PMID:23806648

  10. Structural basis of SETD6-mediated regulation of the NF-kB network via methyl-lysine signaling

    SciTech Connect

    Chang, Yanqi; Levy, Dan; Horton, John R.; Peng, Junmin; Zhang, Xing; Gozani, Or; Cheng, Xiaodong

    2011-10-10

    SET domain containing 6 (SETD6) monomethylates the RelA subunit of nuclear factor kappa B (NF-{kappa}B). The ankyrin repeats of G9a-like protein (GLP) recognizes RelA monomethylated at Lys310. Adjacent to Lys310 is Ser311, a known phosphorylation site of RelA. Ser311 phosphorylation inhibits Lys310 methylation by SETD6 as well as binding of Lys310me1 by GLP. The structure of SETD6 in complex with RelA peptide containing the methylation site, in the presence of S-adenosyl-l-methionine, reveals a V-like protein structure and suggests a model for NF-{kappa}B binding to SETD6. In addition, structural modeling of the GLP ankyrin repeats bound to Lys310me1 peptide provides insight into the molecular basis for inhibition of Lys310me1 binding by Ser311 phosphorylation. Together, these findings provide a structural explanation for a key cellular signaling pathway centered on RelA Lys310 methylation, which is generated by SETD6 and recognized by GLP, and incorporate a methylation-phosphorylation switch of adjacent lysine and serine residues. Finally, SETD6 is structurally similar to the Rubisco large subunit methyltransferase. Given the restriction of Rubisco to plant species, this particular appearance of the protein lysine methyltransferase has been evolutionarily well conserved.

  11. Acetylation of Lysine 201 Inhibits the DNA-Binding Ability of PhoP to Regulate Salmonella Virulence

    PubMed Central

    Tan, Yongcong; Tao, Jing; Ni, Jinjing; Liu, Shuting; Fan, Xia; Zhao, Wei; Lu, Jie; Wu, Wenjuan; Yao, Yu-Feng

    2016-01-01

    The two-component system PhoP-PhoQ is highly conserved in bacteria and regulates virulence in response to various signals for bacteria within the mammalian host. Here, we demonstrate that PhoP could be acetylated by Pat and deacetylated by deacetylase CobB enzymatically in vitro and in vivo in Salmonella Typhimurium. Specifically, the conserved lysine residue 201(K201) in winged helix–turn–helix motif at C-terminal DNA-binding domain of PhoP could be acetylated, and its acetylation level decreases dramatically when bacteria encounter low magnesium, acid stress or phagocytosis of macrophages. PhoP has a decreased acetylation and increased DNA-binding ability in the deletion mutant of pat. However, acetylation of K201 does not counteract PhoP phosphorylation, which is essential for PhoP activity. In addition, acetylation of K201 (mimicked by glutamine substitute) in S. Typhimurium causes significantly attenuated intestinal inflammation as well as systemic infection in mouse model, suggesting that deacetylation of PhoP K201 is essential for Salmonella pathogenesis. Therefore, we propose that the reversible acetylation of PhoP K201 may ensure Salmonella promptly respond to different stresses in host cells. These findings suggest that reversible lysine acetylation in the DNA-binding domain, as a novel regulatory mechanism of gene expression, is involved in bacterial virulence across microorganisms. PMID:26943369

  12. Kdm2a/b Lysine Demethylases Regulate Canonical Wnt Signaling by Modulating the Stability of Nuclear β-Catenin.

    PubMed

    Lu, Lei; Gao, Yan; Zhang, Zan; Cao, Qing; Zhang, Xuena; Zou, Jianghuan; Cao, Ying

    2015-06-22

    In the absence of Wnt activation, cytosolic β-catenin is degraded through GSK3/CK1-mediated phosphorylation at the N terminus. Here, we show that, upon Wnt activation, the stability of nuclear β-catenin is regulated via methylation/demethylation. The protein lysine demethylases Kdm2a and Kdm2b regulate the turnover of non-phosphorylated β-catenin specifically within the nucleus via direct interaction with the fourth and fifth armadillo repeats. The lysine residues within this region are required for the methylation of non-phosphorylated β-catenin, which is demethylated by Kdm2a/b and subsequently ubiquitylated. During Xenopus embryogenesis, kdm2a/b genes are transcribed during early embryogenesis and are required for the specification of the body axis. Kdm2a/b knockdown in Xenopus embryos leads to increases in non-phosphorylated and methylated β-catenin, concurrent with the upregulation of β-catenin target genes. This mechanism is required for controlling the output of the Wnt/β-catenin signaling pathway to maintain normal cellular functions. PMID:26004508

  13. Nuclear magnetic resonance studies of lysine-vasopressin: structural constraints.

    PubMed

    Von Dreele, P H; Brewster, A I; Bovey, F A; Scheraga, H A; Ferger, M F; Du Vigneaud, V

    1971-12-01

    The 220-MHz proton NMR spectra of lysine-vasopressin and some related compounds are examined in deuterated dimethyl sulfoxide to obtain structural information that must be satisfied by any proposed conformation of the molecule. This structural information is in the form of dihedral angles (for rotation about the NH-C(alpha)H bonds) from coupling constants, possible hydrogen bonding of the CONH(2) and backbone amide groups from the temperature-dependence of the chemical shift, and aromatic ring-aromatic ring interaction from the effect of the magnetically anisotropic groups on the chemical shift. PMID:5289251

  14. The conserved lysine of the catalytic domain of protein kinases is actively involved in the phosphotransfer reaction and not required for anchoring ATP.

    PubMed Central

    Carrera, A C; Alexandrov, K; Roberts, T M

    1993-01-01

    The study of the various protein kinases reveals that, despite their considerably diversity, they have evolved from a common origin. Eleven conserved subdomains have been described that encompass the catalytic core of these enzymes. One of these conserved regions, subdomain II, contains an invariant lysine residue present in all known protein kinase catalytic domains. Two facts have suggested that this conserved lysine of subdomain II is essential for binding ATP: (i) several investigators have demonstrated that this residue is physically proximal to the ATP molecule, and (ii) conservative substitutions at this site render the kinase inactive. However, these results are also consistent with a functional role of the conserved lysine of subdomain II in orienting or facilitating the transfer of phosphate. To study in more detail the role of subdomain II, we have generated mutants of the protein-tyrosine kinase pp56lck that have single amino acid substitutions within the area surrounding the conserved residue Lys-273 in subdomain II. When compared with wild-type pp56lck, these mutants displayed profound reductions in their phosphotransfer efficiencies and small differences in their affinities for ATP. Further, the substitution of arginine for Lys-273 resulted in a mutant protein unable to transfer the gamma-phosphate of ATP but able to bind 8-azido-ATP with an efficiency similar to that of wild-type pp56lck. These results suggest that the region including Lys-273 of subdomain II is involved in the enzymatic process of phosphate transfer, rather than in anchoring ATP. Images PMID:8421674

  15. NKLP27: A Teleost NK-Lysin Peptide that Modulates Immune Response, Induces Degradation of Bacterial DNA, and Inhibits Bacterial and Viral Infection

    PubMed Central

    Sun, Li

    2014-01-01

    NK-lysin is an antimicrobial protein produced by cytotoxic T lymphocytes and natural killer cells. In this study, we examined the biological property of a peptide, NKLP27, derived from tongue sole (Cynoglossus semilaevis) NK-lysin. NKLP27 is composed of 27 amino acids and shares little sequence identity with known NK-lysin peptides. NKLP27 possesses bactericidal activity against both Gram-negative and Gram-positive bacteria including common aquaculture pathogens. The bactericidal activity of NKLP27 was dependent on the C-terminal five residues, deletion of which dramatically reduced the activity of NKLP27. During its interaction with the target bacterial cells, NKLP27 destroyed cell membrane integrity, penetrated into the cytoplasm, and induced degradation of genomic DNA. In vivo study showed that administration of tongue sole with NKLP27 before bacterial and viral infection significantly reduced pathogen dissemination and replication in tissues. Further study revealed that fish administered with NKLP27 exhibited significantly upregulated expression of the immune genes including those that are known to be involved in antibacterial and antiviral defense. These results indicate that NKLP27 is a novel antimicrobial against bacterial and viral pathogens, and that the observed effect of NKLP27 on bacterial DNA and host gene expression adds new insights to the action mechanism of fish antimicrobial peptides. PMID:25180858

  16. Amino-acid selective experiments on uniformly 13C and 15N labeled proteins by MAS NMR: Filtering of lysines and arginines

    NASA Astrophysics Data System (ADS)

    Jehle, Stefan; Rehbein, Kristina; Diehl, Anne; van Rossum, Barth-Jan

    2006-12-01

    Amino-acid selective magic-angle spinning (MAS) NMR experiments can aid the assignment of ambiguous cross-peaks in crowded spectra of solid proteins. In particular for larger proteins, data analysis can be hindered by severe resonance overlap. In such cases, filtering techniques may provide a good alternative to site-specific spin-labeling to obtain unambiguous assignments that can serve as starting points in the assignment procedure. In this paper we present a simple pulse sequence that allows selective excitation of arginine and lysine residues. To achieve this, we make use of a combination of specific cross-polarization for selective excitation [M. Baldus, A.T. Petkova, J. Herzfeld, R.G. Griffin, Cross polarization in the tilted frame: assignment and spectral simplification in heteronuclear spin systems, Mol. Phys. 95 (1998) 1197-1207.] and spin diffusion for transfer along the amino-acid side-chain. The selectivity of the filter is demonstrated with the excitation of lysine and arginine side-chain resonances in a uniformly 13C and 15N labeled protein preparation of the α-spectrin SH3 domain. It is shown that the filter can be applied as a building block in a 13C- 13C lysine-only correlation experiment.

  17. Structural Basis for Lower Lysine Methylation State-Specific Readout by MBT Repeats of L3MBTL1 and an Engineered PHD Finger

    SciTech Connect

    Li, Haitao; Fischle, Wolfgang; Wang, Wooikoon; Duncan, Elizabeth M.; Liang, Lena; Murakami-Ishibe, Satoko; Allis, C. David; Patel, Dinshaw J.

    2008-09-17

    Human L3MBTL1, which contains three malignant brain tumor (MBT) repeats, binds monomethylated and dimethylated lysines, but not trimethylated lysines, in several histone sequence contexts. In crystal structures of L3MBTL1 complexes, the monomethyl- and dimethyllysines insert into a narrow and deep cavity of aromatic residue-lined pocket 2, while a proline ring inserts into shallower pocket 1. We have also engineered a single Y to E substitution within the aromatic cage of the BPTF PHD finger, resulting in a reversal of binding preference from trimethyl- to dimethyllysine in an H3K4 sequence context. In both the 'cavity insertion' (L3MBTL1) and 'surface groove' (PHD finger) modes of methyllysine recognition, a carboxylate group both hydrogen bonds and ion pairs to the methylammonium proton. Our structural and binding studies of these two modules provide insights into the molecular principles governing the decoding of lysine methylation states, thereby highlighting a methylation state-specific layer of histone mark readout impacting on epigenetic regulation.

  18. Bacteriophage lysin mediates the binding of streptococcus mitis to human platelets through interaction with fibrinogen.

    PubMed

    Seo, Ho Seong; Xiong, Yan Q; Mitchell, Jennifer; Seepersaud, Ravin; Bayer, Arnold S; Sullam, Paul M

    2010-01-01

    The binding of bacteria to human platelets is a likely central mechanism in the pathogenesis of infective endocarditis. We have previously found that platelet binding by Streptococcus mitis SF100 is mediated by surface components encoded by a lysogenic bacteriophage, SM1. We now demonstrate that SM1-encoded lysin contributes to platelet binding via its direct interaction with fibrinogen. Far Western blotting of platelets revealed that fibrinogen was the major membrane-associated protein bound by lysin. Analysis of lysin binding with purified fibrinogen in vitro confirmed that these proteins could bind directly, and that this interaction was both saturable and inhibitable. Lysin bound both the Aalpha and Bbeta chains of fibrinogen, but not the gamma subunit. Binding of lysin to the Bbeta chain was further localized to a region within the fibrinogen D fragment. Disruption of the SF100 lysin gene resulted in an 83+/-3.1% reduction (mean +/- SD) in binding to immobilized fibrinogen by this mutant strain (PS1006). Preincubation of this isogenic mutant with purified lysin restored fibrinogen binding to wild type levels. When tested in a co-infection model of endocarditis, loss of lysin expression resulted in a significant reduction in virulence, as measured by achievable bacterial densities (CFU/g) within vegetations, kidneys, and spleens. These results indicate that bacteriophage-encoded lysin is a multifunctional protein, representing a new class of fibrinogen-binding proteins. Lysin appears to be cell wall-associated through its interaction with choline. Once on the bacterial surface, lysin can bind fibrinogen directly, which appears to be an important interaction for the pathogenesis of endocarditis. PMID:20714354

  19. The fermentative production of L-lysine as an animal feed additive.

    PubMed

    Kircher, M; Pfefferle, W

    2001-04-01

    A new and innovative process for the biotechnological production of L-lysine is presented, exemplified here by the fermentative production of the feed additive Biolys60. The novel feature of this product is that the entire manufacturing concept, i.e. the production strain, the raw materials, all process stages and the product specifications have been systematically tailored for optimal environmental compatibility and for minimum resource depletion and waste. The process completely dispenses with the need to discharge residual and waste material and reduces the handling of hazardous materials to a minimum. Since only a few process stages are involved, the method is economical to use and investment outlay is reduced. The process, which also leads to a higher grade product, is thus highly attractive in both ecological and economical terms. By boosting the nutrient value of the plant-based feedstuffs, the product itself makes an cost-effective contribution towards a more sustainable form of animal feeding and by reducing nitrogen emission levels promotes a more environmentally compatible form of animal husbandry. PMID:11233822

  20. Regulation of Estrogen Receptor [alpha] by the SET7 Lysine Methyltransferase

    SciTech Connect

    Subramanian, Krithika; Jia, Da; Kapoor-Vazirani, Priya; Powell, Doris R.; Collins, Robert E.; Sharma, Dipali; Peng, Junmin; Cheng, Xiaodong; Vertino, Paula M.

    2008-09-12

    Estrogen receptor {alpha} (ER) is a ligand-dependent transcription factor. Upon binding estrogen, ER recruits coactivator complexes with histone acetyltransferase or methyltransferase activities to activate downstream target genes. In addition to histones, coactivators can modify ER itself and other proteins in the transactivation complex. Here, we show that ER is directly methylated at lysine 302 (K302) by the SET7 methyltransferase. SET7-mediated methylation stabilizes ER and is necessary for the efficient recruitment of ER to its target genes and for their transactivation. The SET7-ER complex structure reveals the molecular basis for ER peptide recognition and predicts that modifications or mutations of nearby residues would affect K302 methylation. Indeed, a breast cancer-associated mutation at K303 (K303R) alters methylation at K302 in vitro and in vivo. These findings raise the possibility that generation, recognition, and removal of modifications within the ER hinge region generate 'ER modification cassettes' that yield distinct patterns for signaling downstream events.

  1. Crystal Structure and Functional Analysis of Homocitrate Synthase, an Essential Enzyme in Lysine Biosynthesis

    SciTech Connect

    Bulfer, Stacie L.; Scott, Erin M.; Couture, Jean-François; Pillus, Lorraine; Trievel, Raymond C.

    2010-01-12

    Homocitrate synthase (HCS) catalyzes the first and committed step in lysine biosynthesis in many fungi and certain Archaea and is a potential target for antifungal drugs. Here we report the crystal structure of the HCS apoenzyme from Schizosaccharomyces pombe and two distinct structures of the enzyme in complex with the substrate 2-oxoglutarate (2-OG). The structures reveal that HCS forms an intertwined homodimer stabilized by domain-swapping between the N- and C-terminal domains of each monomer. The N-terminal catalytic domain is composed of a TIM barrel fold in which 2-OG binds via hydrogen bonds and coordination to the active site divalent metal ion, whereas the C-terminal domain is composed of mixed {alpha}/{beta} topology. In the structures of the HCS apoenzyme and one of the 2-OG binary complexes, a lid motif from the C-terminal domain occludes the entrance to the active site of the neighboring monomer, whereas in the second 2-OG complex the lid is disordered, suggesting that it regulates substrate access to the active site through its apparent flexibility. Mutations of the active site residues involved in 2-OG binding or implicated in acid-base catalysis impair or abolish activity in vitro and in vivo. Together, these results yield new insights into the structure and catalytic mechanism of HCSs and furnish a platform for developing HCS-selective inhibitors.

  2. Diatom Mimics: Directing the Formation of Biosilica Nanoparticles by Controlled Folding of Lysine-Leucine Peptides

    PubMed Central

    Baio, Joe E.; Zane, Ariel; Jaeger, Vance; Roehrich, Adrienne M.; Lutz, Helmut; Pfaendtner, Jim; Drobny, Gary P.; Weidner, Tobias

    2015-01-01

    Silaffins, long chain polyamines, and other biomolecules found in diatoms are involved in the assembly of a large number of silica nanostructures under mild, ambient conditions. Nanofabrication researchers have sought to mimic the diatom's biosilica production capabilities by engineering proteins to resemble aspects of naturally occurring biomolecules. Such mimics can produce monodisperse biosilica nanospheres, but in vitro production of the variety of intricate biosilica nanostructures that compose the diatom frustule is not yet possible. In this study we demonstrate how LK peptides, composed solely of lysine (K) and leucine (L) amino acids arranged with varying hydrophobic periodicities, initiate the formation of different biosilica nanostructures in vitro. When L and K residues are arranged with a periodicity of 3.5 the α-helical form of the LK peptide produces monodisperse biosilica nanospheres. However, when the LK periodicity is changed to 3.0, corresponding to a 310 helix, the morphology of the nanoparticles changes to elongated rod-like structures. β-strand LK peptides with a periodicity of 2.0 induce wire-like silica morphologies. This study illustrates how the morphology of biosilica can be changed simply by varying the periodicity of polar and nonpolar amino acids. PMID:25285787

  3. Diatom mimics: directing the formation of biosilica nanoparticles by controlled folding of lysine-leucine peptides.

    PubMed

    Baio, Joe E; Zane, Ariel; Jaeger, Vance; Roehrich, Adrienne M; Lutz, Helmut; Pfaendtner, Jim; Drobny, Gary P; Weidner, Tobias

    2014-10-29

    Silaffins, long chain polyamines, and other biomolecules found in diatoms are involved in the assembly of a large number of silica nanostructures under mild, ambient conditions. Nanofabrication researchers have sought to mimic the diatom's biosilica production capabilities by engineering proteins to resemble aspects of naturally occurring biomolecules. Such mimics can produce monodisperse biosilica nanospheres, but in vitro production of the variety of intricate biosilica nanostructures that compose the diatom frustule is not yet possible. In this study we demonstrate how LK peptides, composed solely of lysine (K) and leucine (L) amino acids arranged with varying hydrophobic periodicities, initiate the formation of different biosilica nanostructures in vitro. When L and K residues are arranged with a periodicity of 3.5 the α-helical form of the LK peptide produces monodisperse biosilica nanospheres. However, when the LK periodicity is changed to 3.0, corresponding to a 310 helix, the morphology of the nanoparticles changes to elongated rod-like structures. β-strand LK peptides with a periodicity of 2.0 induce wire-like silica morphologies. This study illustrates how the morphology of biosilica can be changed simply by varying the periodicity of polar and nonpolar amino acids. PMID:25285787

  4. Lysines 3241 and 3260 of DNA-PKcs are important for genomic stability and radioresistance.

    PubMed

    Mori, Eiichiro; Davis, Anthony J; Hasegawa, Masatoshi; Chen, David J

    2016-08-19

    DNA-dependent protein kinase (DNA-PK) is a serine/threonine kinase that plays an essential role in the repair of DNA double-strand breaks (DSBs) in the non-homologous end-joining (NHEJ) pathway. The DNA-PK holoenzyme consists of a catalytic subunit (DNA-PKcs) and DNA-binding subunit (Ku70/80, Ku). Ku is a molecular sensor for double-stranded DNA and once bound to DSB ends it recruits DNA-PKcs to the DSB site. Subsequently, DNA-PKcs is activated and heavily phosphorylated, with these phosphorylations modulating DNA-PKcs. Although phosphorylation of DNA-PKcs is well studied, other post-translational modifications of DNA-PKcs are not. In this study, we aimed to determine if acetylation of DNA-PKcs regulates DNA-PKcs-dependent DSB repair. We report that DNA-PKcs is acetylated in vivo and identified two putative acetylation sites, lysine residues 3241 and 3260. Mutating these sites to block potential acetylation results in increased radiosensitive, a slight decrease in DSB repair capacity as assessed by γH2AX resolution, and increased chromosomal aberrations, especially quadriradial chromosomes. Together, our results provide evidence that acetylation potentially regulates DNA-PKcs. PMID:27297111

  5. Application of the MIDAS approach for analysis of lysine acetylation sites.

    PubMed

    Evans, Caroline A; Griffiths, John R; Unwin, Richard D; Whetton, Anthony D; Corfe, Bernard M

    2013-01-01

    Multiple Reaction Monitoring Initiated Detection and Sequencing (MIDAS™) is a mass spectrometry-based technique for the detection and characterization of specific post-translational modifications (Unwin et al. 4:1134-1144, 2005), for example acetylated lysine residues (Griffiths et al. 18:1423-1428, 2007). The MIDAS™ technique has application for discovery and analysis of acetylation sites. It is a hypothesis-driven approach that requires a priori knowledge of the primary sequence of the target protein and a proteolytic digest of this protein. MIDAS essentially performs a targeted search for the presence of modified, for example acetylated, peptides. The detection is based on the combination of the predicted molecular weight (measured as mass-charge ratio) of the acetylated proteolytic peptide and a diagnostic fragment (product ion of m/z 126.1), which is generated by specific fragmentation of acetylated peptides during collision induced dissociation performed in tandem mass spectrometry (MS) analysis. Sequence information is subsequently obtained which enables acetylation site assignment. The technique of MIDAS was later trademarked by ABSciex for targeted protein analysis where an MRM scan is combined with full MS/MS product ion scan to enable sequence confirmation. PMID:23381851

  6. Regulation of Estrogen Receptor Alpha by the SET7 lysine methyltransferase

    PubMed Central

    Subramanian, Krithika; Jia, Da; Kapoor-Vazirani, Priya; Powell, Doris R.; Collins, Robert E.; Sharma, Dipali; Peng, Junmin; Cheng, Xiaodong; Vertino, Paula M.

    2008-01-01

    Summary Estrogen receptor α (ER) is a ligand-dependent transcription factor. Upon binding estrogen, ER recruits coactivator complexes with histone acetyltransferase or methyltransferase activities to activate downstream target genes. In addition to histones, coactivators can modify ER itself and other proteins in the transactivation complex. Here, we show that ER is directly methylated at lysine 302 (K302) by the SET7 methyltransferase. SET7-mediated methylation stabilizes ER and is necessary for the efficient recruitment of ER to its target genes, and their transactivation. The SET7-ER complex structure reveals the molecular basis for ER peptide recognition and predicts that modifications or mutations of nearby residues would affect K302 methylation. Indeed, a breast cancer-associated mutation at K303 (K303R) alters methylation at K302 in vitro and in vivo. These findings raise the possibility that generation, recognition, and removal of modifications within the ER hinge region generates “ER modification cassettes” that yield distinct patterns for signaling downstream events. PMID:18471979

  7. Kinetics of N(ε)-(carboxymethyl)lysine formation in aqueous model systems of sugars and casein.

    PubMed

    Nguyen, Ha T; van der Fels-Klerx, H J; van Boekel, Martinus A J S

    2016-02-01

    This study investigated the formation of N(ε)-carboxymethyllysine (CML) in two caseinate solutions containing: (1) glucose, (2) lactose, each heated at 120 °C and 130 °C. At both heating temperatures, CML concentration in lactose-caseinate solution was higher than in glucose-caseinate solution. In both solutions, more CML was formed at 130 °C than at 120 °C. Using multiresponse modelling, two degradation routes for the sugars were confirmed: (1) isomerisation of glucose or lactose and subsequent degradation via Lobry de Bruyn-Alberda van Ekenstein (LA) arrangement; (2) the Maillard reaction between the reducing sugar and lysine residues. Modelling results suggested that CML was not formed from oxidation of the reducing sugars, but from the Maillard reaction via the Amadori rearrangement product. Since CML appeared to be thermally unstable under the current study conditions, it may not be a perfect indicator for heat damage of processed foods. This is the first study in which CML formation was linked to available information on the Maillard reaction via multiresponse modelling. PMID:26304329

  8. The Mycobacterium tuberculosis LipB enzyme functions as a cysteine/lysine dyad acyltransferase

    PubMed Central

    Ma, Qingjun; Zhao, Xin; Eddine, Ali Nasser; Geerlof, Arie; Li, Xinping; Cronan, John E.; Kaufmann, Stefan H. E.; Wilmanns, Matthias

    2006-01-01

    Lipoic acid is essential for the activation of a number of protein complexes involved in key metabolic processes. Growth of Mycobacterium tuberculosis relies on a pathway in which the lipoate attachment group is synthesized from an endogenously produced octanoic acid moiety. In patients with multiple-drug-resistant M. tuberculosis, expression of one gene from this pathway, lipB, encoding for octanoyl-[acyl carrier protein]-protein acyltransferase is considerably up-regulated, thus making it a potential target in the search for novel antiinfectives against tuberculosis. Here we present the crystal structure of the M. tuberculosis LipB protein at atomic resolution, showing an unexpected thioether-linked active-site complex with decanoic acid. We provide evidence that the transferase functions as a cysteine/lysine dyad acyltransferase, in which two invariant residues (Lys-142 and Cys-176) are likely to function as acid/base catalysts. Analysis by MS reveals that the LipB catalytic reaction proceeds by means of an internal thioesteracyl intermediate. Structural comparison of LipB with lipoate protein ligase A indicates that, despite conserved structural and sequence active-site features in the two enzymes, 4′-phosphopantetheine-bound octanoic acid recognition is a specific property of LipB. PMID:16735476

  9. Peptide Nucleic Acid with a Lysine Side Chain at the β-Position: Synthesis and Application for DNA Cleavage.

    PubMed

    Sugiyama, Toru; Kuwata, Keiko; Imamura, Yasutada; Demizu, Yosuke; Kurihara, Masaaki; Takano, Masashi; Kittaka, Atsushi

    2016-01-01

    This paper reports the synthesis of new β-Lys peptide nucleic acid (PNA) monomers and their incorporation into a 10-residue PNA sequence. PNA containing β-Lys PNA units formed a stable hybrid duplex with DNA. However, incorporation of β-Lys PNA units caused destabilization of PNA-DNA duplexes to some extent. Electrostatic attractions between β-PNA and DNA could reduce this destabilization effect. Subsequently, bipyridine-conjugated β-Lys PNA was prepared and exhibited sequence selective cleavage of DNA. Based on the structures of the cleavage products and molecular modeling, we reasoned that bipyridine moiety locates within the minor groove of the PNA-DNA duplexes. The lysine side chain of β-PNA is a versatile handle for attaching various functional molecules. PMID:27373637

  10. Michael addition of dehydroalanine-containing MAPK peptides to catalytic lysine inhibits the activity of phosphothreonine lyase.

    PubMed

    Zhang, Yuan; Yang, Ru; Huang, Juan; Liang, Qiujin; Guo, Yanmin; Bian, Weixiang; Luo, Lingfei; Li, Hongtao

    2015-11-30

    The phosphothreonine lyases OspF and SpvC irreversibly inactivate host dual-phosphorylated mitogen-activated protein kinases (MAPKs) [pThr-X-pTyr motif] through β-elimination. We found that dual-phosphorylated (pSer-X-pTyr) MAPK substrate peptides and their resulting catalytic products cross-link to OspF and SpvC. Mass spectrometry results revealed that these linkages form between lysine, which acts as a general base, and dehydroalanine (Dha) on catalytic products. The nucleophilic addition efficiency is dependent on the K136 residue being in a deprotonated state. Peptide cross-linking inhibits the activity of SpvC and blocks the inactivation of MAPK signaling by SpvC. Small compounds mimicking these sequences may act as phosphothreonine lyase inhibitors. PMID:26519561

  11. Determination of Solubility Parameters of Ibuprofen and Ibuprofen Lysinate.

    PubMed

    Kitak, Teja; Dumičić, Aleksandra; Planinšek, Odon; Šibanc, Rok; Srčič, Stanko

    2015-01-01

    In recent years there has been a growing interest in formulating solid dispersions, which purposes mainly include solubility enhancement, sustained drug release and taste masking. The most notable problem by these dispersions is drug-carrier (in)solubility. Here we focus on solubility parameters as a tool for predicting the solubility of a drug in certain carriers. Solubility parameters were determined in two different ways: solely by using calculation methods, and by experimental approaches. Six different calculation methods were applied in order to calculate the solubility parameters of the drug ibuprofen and several excipients. However, we were not able to do so in the case of ibuprofen lysinate, as calculation models for salts are still not defined. Therefore, the extended Hansen's approach and inverse gas chromatography (IGC) were used for evaluating of solubility parameters for ibuprofen lysinate. The obtained values of the total solubility parameter did not differ much between the two methods: by the extended Hansen's approach it was δt = 31.15 MPa(0.5) and with IGC it was δt = 35.17 MPa(0.5). However, the values of partial solubility parameters, i.e., δd, δp and δh, did differ from each other, what might be due to the complex behaviour of a salt in the presence of various solvents. PMID:26633347

  12. Evolution of a novel lysine decarboxylase in siderophore biosynthesis.

    PubMed

    Burrell, Matthew; Hanfrey, Colin C; Kinch, Lisa N; Elliott, Katherine A; Michael, Anthony J

    2012-10-01

    Structural backbones of iron-scavenging siderophore molecules include polyamines 1,3-diaminopropane and 1,5-diaminopentane (cadaverine). For the cadaverine-based desferroxiamine E siderophore in Streptomyces coelicolor, the corresponding biosynthetic gene cluster contains an ORF encoded by desA that was suspected of producing the cadaverine (decarboxylated lysine) backbone. However, desA encodes an l-2,4-diaminobutyrate decarboxylase (DABA DC) homologue and not any known form of lysine decarboxylase (LDC). The only known function of DABA DC is, together with l-2,4-aminobutyrate aminotransferase (DABA AT), to synthesize 1,3-diaminopropane. We show here that S. coelicolor desA encodes a novel LDC and we hypothesized that DABA DC homologues present in siderophore biosynthetic clusters in the absence of DABA AT ORFs would be novel LDCs. We confirmed this by correctly predicting the LDC activity of a DABA DC homologue from a Yersinia pestis siderophore biosynthetic pathway. The corollary was confirmed for a DABA DC homologue, adjacent to a DABA AT ORF in a siderophore pathway in the cyanobacterium Anabaena variabilis, which was shown to be a bona fide DABA DC. These findings enable prediction of whether a siderophore pathway will utilize 1,3-diaminopropane or cadaverine, and suggest that the majority of bacteria use DABA AT and DABA DC for siderophore, rather than norspermidine/polyamine biosynthesis. PMID:22906379

  13. Exposure of the lysine in the gamma chain dodecapeptide of human fibrinogen is not enhanced by adsorption to poly(ethylene terephthalate) as measured by biotinylation and mass spectrometry.

    PubMed

    Ovod, Vitaliy; Scott, Evan A; Flake, Megan M; Parker, Stanley R; Bateman, Randall J; Elbert, Donald L

    2012-03-01

    Conformational changes in adsorbed fibrinogen may enhance the exposure of platelet adhesive sites that are inaccessible in solution. To test this hypothesis, mass spectrometric methods were developed to quantify chemical modification of lysine residues following adsorption of fibrinogen to biomaterials. The quantitative method used an internal standard consisting of isotope-labeled fibrinogen secreted by human HepG2 cells in culture. Lysine residues in the internal standard were partially reacted with NHS-biotin. For the experimental samples, normal human fibrinogen was adsorbed to poly(ethylene terephthalate) (PET) particles. The adsorbed fibrinogen was reacted with NHS-biotin and then eluted from the particles. Constant amounts of internal standard were added to sample fibrinogen and analyzed by liquid chromatography/tandem mass spectrometry. Biotinylation of the lysine residue in the platelet-adhesive gamma chain dodecapeptide (GCDP) was quantified by comparison with the internal standard. Approximately 80% of the GCDP peptides were biotinylated when fibrinogen was reacted with NHS-biotin in solution or adsorbed onto PET. These results are generally consistent with previous antibody binding studies and suggest that other regions of fibrinogen may be crucial in promoting platelet adhesion to materials. The results do not directly address but are consistent with the hypothesis that only activated platelets adhere to adsorbed fibrinogen. PMID:22213354

  14. Improving lysine production by Corynebacterium glutamicum through DNA microarray-based identification of novel target genes.

    PubMed

    Sindelar, Georg; Wendisch, Volker F

    2007-09-01

    For the biotechnological production of L: -lysine, mainly strains of Corynebacterium glutamicum are used, which have been obtained by classical mutagenesis and screening or selection or by metabolic engineering. Gene targets for the amplification and deregulation of the lysine biosynthesis pathway, for the improvement of carbon precursor supply and of nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH) regeneration, are known. To identify novel target genes to improve lysine production, the transcriptomes of the classically obtained lysine producing strain MH20-22B and several other C. glutamicum strains were compared. As lysine production by the classically obtained strain, which possesses feedback-resistant aspartokinase and is leucine auxotrophic, exceeds that of a genetically defined leucine auxotrophic wild-type derivative possessing feedback-resistant aspartokinase, additional traits beneficial for lysine production are present. NCgl0855, putatively encoding a methyltransferase, and the amtA-ocd-soxA operon, encoding an ammonium uptake system, a putative ornithine cyclodeaminase and an uncharacterized enzyme, were among the genes showing increased expression in the classically obtained strain irrespective of the presence of feedback-resistant aspartokinase. Lysine production could be improved by about 40% through overexpression of NCgl0855 or the amtA-ocd-soxA operon. Thus, novel target genes for the improvement of lysine production could be identified in a discovery-driven approach based on global gene expression analysis. PMID:17364200

  15. Differential lysine acetylation profiles of Erwinia amylovora strains revealed by proteomics

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein lysine acetylation (LysAc) in bacteria has recently been demonstrated to be widespread in E. coli and Salmonella and to broadly regulate bacterial physiology and metabolism. However, LysAc in plant pathogenic bacteria is largely unknown. Here we report the lysine acetylome of Erwinia amylovo...

  16. Lysine Acetylation Is a Widespread Protein Modification for Diverse Proteins in Arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lysine acetylation (LysAc), a form of reversible protein post translational modification previously known only for histone proteins in plants, is shown to be wide spread in Arabidopsis. Sixty five lysine modification sites were identified on 58 proteins, which operate in a wide variety of pathways/...

  17. Carnitine biosynthesis in Neurospora crassa: enzymatic conversion of lysine to epsilon-N-trimethyllysine.

    PubMed Central

    Rebouche, C J; Broquist, H P

    1976-01-01

    The enzymatic conversion of L-lysine, epsilon-N-trimethyl-L-lysine the first series of reactions in the biosynthesis of carnitine in Neurospora crassa, proceeds via sequential methylation of free L-lysine, epsilon-N-methyl-L-lysine, and epsilon -N-dimethyl-L-lysine. The latter two compounds have been shown to be intermediates in the biosynthesis of carnitine by radioisotope dilution and incorporation experiments in growing cultures of N. crassa 33933 (lys-) and 38706 (met-). Methionine but not choline, has been recognized as an effective methyl donor in vivo. Inclusion of choline in the growth medium of strain 33933 does, however, enhance incorporation of the methyl groups of L-[methyl-3H]methionine into carnitine in an apparent "sparing" effect on methionine synthesis. Studies in cell-free extracts of the lysine auxotroph strain 33933 of N. crassa have established that lysine and epsilon-N-methyl and epsilon-N-dimethyllysine are enzymatically methylated, with S-adenosyl-L-methionine as the methyl group donor. The enzyme system appears to have no essential cofactors. Lysine does not induce synthesis of the enzyme system in the wild-type strain 262, whereas both carnitine and epsilon-N-trimethyllysine repress its synthesis in strain 33933. PMID:133101

  18. Global Profiling of Protein Lysine Malonylation in Escherichia coli Reveals Its Role in Energy Metabolism.

    PubMed

    Qian, Lili; Nie, Litong; Chen, Ming; Liu, Ping; Zhu, Jun; Zhai, Linhui; Tao, Sheng-Ce; Cheng, Zhongyi; Zhao, Yingming; Tan, Minjia

    2016-06-01

    Protein lysine malonylation is a recently identified post-translational modification (PTM), which is evolutionarily conserved from bacteria to mammals. Although analysis of lysine malonylome in mammalians suggested that this modification was related to energy metabolism, the substrates and biological roles of malonylation in prokaryotes are still poorly understood. In this study, we performed qualitative and quantitative analyses to globally identify lysine malonylation substrates in Escherichia coli. We identified 1745 malonylation sites in 594 proteins in E. coli, representing the first and largest malonylome data set in prokaryotes up to date. Bioinformatic analyses showed that lysine malonylation was significantly enriched in protein translation, energy metabolism pathways and fatty acid biosynthesis, implying the potential roles of protein malonylation in bacterial physiology. Quantitative proteomics by fatty acid synthase inhibition in both auxotrophic and prototrophic E. coli strains revealed that lysine malonylation is closely associated with E. coli fatty acid metabolism. Protein structural analysis and mutagenesis experiment suggested malonylation could impact enzymatic activity of citrate synthase, a key enzyme in citric acid (TCA) cycle. Further comparative analysis among lysine malonylome, succinylome and acetylome data showed that these three modifications could participate in some similar enriched metabolism pathways, but they could also possibly play distinct roles such as in fatty acid synthesis. These data expanded our knowledge of lysine malonylation in prokaryotes, providing a resource for functional study of lysine malonylation in bacteria. PMID:27183143

  19. New enzymatic methods for selective assay of L-lysine using an L-lysine specific decarboxylase/oxidase from Burkholderia sp. AIU 395.

    PubMed

    Sugawara, Asami; Matsui, Daisuke; Yamada, Miwa; Asano, Yasuhisa; Isobe, Kimiyasu

    2015-03-01

    We developed new enzymatic methods for the selective assay of L-lysine by utilizing an oxidase reaction and a decarboxylation reaction by the L-lysine-specific decarboxylase/oxidase (L-Lys-DC/OD) from Burkholderia sp. AIU 395. The method utilizing the oxidase reaction of this enzyme was useful for determination of high concentrations of L-lysine. The method utilizing the decarboxylase reaction, which proceeded via the combination of the L-Lys-DC/OD and putrescine oxidase (PUO) from Micrococcus rubens, was effective for determination of low concentrations of L-lysine. Both methods showed good linearity, and neither was affected by other amino acids or amines. In addition, the within-assay and between-assay precisions of both methods were within the allowable range. The coupling of L-Lys-DC/OD with PUO was also useful for the differential assay of L-lysine and cadaverine. These newly developed methods were applied to the assay of L-lysine in biological samples and found to be effective. PMID:25282636

  20. Effect of irradiation on Nε-carboxymethyl-lysine and Nε-carboxyethyl-lysine formation in cooked meat products during storage

    NASA Astrophysics Data System (ADS)

    Yu, Ligang; He, Zhiyong; Zeng, Maomao; Zheng, Zongping; Chen, Jie

    2016-03-01

    This study investigated the effects of irradiation on Nε-carboxymethyl-lysine (CML) and Nε-carboxyethyl-lysine (CEL) formation in cooked red and white meats during storage. The results showed that irradiation did not affect CML/CEL formation (0 weeks). After 6 weeks, CML/CEL contents in the irradiated samples exhibited a higher growth rate than the non-irradiated samples, especially the red meat. The results of electron spin resonance spectrometry and 2-Thiobarbituric acid-reactive substances suggested irradiation had induced free-radical reactions and accelerated lipid oxidation during storage. A linear correlation (r=0.810-0.906, p<0.01) was found between the loss of polyunsaturated fatty acids content and increase of CML/CEL content in the irradiated samples after 0 and 6 weeks of storage. The results indicate that irradiation-induced lipid oxidation promotes CML/CEL formation, and CML/CEL formation by the lipid oxidation pathways may be an important pathway for CML/CEL accumulation in irradiated meat products during storage.

  1. Acetylome analysis reveals the involvement of lysine acetylation in diverse biological processes in Phytophthora sojae.

    PubMed

    Li, Delong; Lv, Binna; Tan, Lingling; Yang, Qianqian; Liang, Wenxing

    2016-01-01

    Lysine acetylation is a dynamic and highly conserved post-translational modification that plays an important regulatory role in almost every aspects of cell metabolism in both eukaryotes and prokaryotes. Phytophthora sojae is one of the most important plant pathogens due to its huge economic impact. However, to date, little is known about the functions of lysine acetylation in this Phytopthora. Here, we conducted a lysine acetylome in P. sojae. Overall, 2197 lysine acetylation sites in 1150 proteins were identified. The modified proteins are involved in diverse biological processes and are localized to multiple cellular compartments. Importantly, 7 proteins involved in the pathogenicity or the secretion pathway of P. sojae were found to be acetylated. These data provide the first comprehensive view of the acetylome of P. sojae and serve as an important resource for functional analysis of lysine acetylation in plant pathogens. PMID:27412925

  2. Purification of L-lysine in simulated moving bed and fixed-bed chromatography.

    PubMed

    Robatjazi, Seyed Mortaza; Shojaosadati, Seyed Abbas; Karbasy, Seyed Mojtaba

    2004-07-01

    L-Lysine was produced by a microbial process utilizing a Corynebacterium glutamicum (ATCC 21799) strain. L-Lysine was purified from the cultivated medium by fixed-bed and simulated moving bed (SMB) chromatography. The separation conditions including pH, eluent concentration and Lys+ and Lys2+ adsorption isotherms were studied in batch adsorption. The column capacity, eluent flow rate and eluent concentration have been studied in fixed-bed chromatography. Maximum purification rate of lysine was obtained as 0.066 g/(g x h) (per gram resin and per hour) at an eluent flow rate of 10 mL/min in fixed-bed chromatography. The results obtained from SMB were 0.11 g/(g x h) for L-lysine purification rate and 96% for L-lysine recovery. PMID:15709427

  3. Smyd2 controls cytoplasmic lysine methylation of Hsp90 and myofilament organization

    PubMed Central

    Donlin, Laura T.; Andresen, Christian; Just, Steffen; Rudensky, Eugene; Pappas, Christopher T.; Kruger, Martina; Jacobs, Erica Y.; Unger, Andreas; Zieseniss, Anke; Dobenecker, Marc-Werner; Voelkel, Tobias; Chait, Brian T.; Gregorio, Carol C.; Rottbauer, Wolfgang; Tarakhovsky, Alexander; Linke, Wolfgang A.

    2012-01-01

    Protein lysine methylation is one of the most widespread post-translational modifications in the nuclei of eukaryotic cells. Methylated lysines on histones and nonhistone proteins promote the formation of protein complexes that control gene expression and DNA replication and repair. In the cytoplasm, however, the role of lysine methylation in protein complex formation is not well established. Here we report that the cytoplasmic protein chaperone Hsp90 is methylated by the lysine methyltransferase Smyd2 in various cell types. In muscle, Hsp90 methylation contributes to the formation of a protein complex containing Smyd2, Hsp90, and the sarcomeric protein titin. Deficiency in Smyd2 results in the loss of Hsp90 methylation, impaired titin stability, and altered muscle function. Collectively, our data reveal a cytoplasmic protein network that employs lysine methylation for the maintenance and function of skeletal muscle. PMID:22241783

  4. Acetylome analysis reveals the involvement of lysine acetylation in diverse biological processes in Phytophthora sojae

    PubMed Central

    Li, Delong; Lv, Binna; Tan, Lingling; Yang, Qianqian; Liang, Wenxing

    2016-01-01

    Lysine acetylation is a dynamic and highly conserved post-translational modification that plays an important regulatory role in almost every aspects of cell metabolism in both eukaryotes and prokaryotes. Phytophthora sojae is one of the most important plant pathogens due to its huge economic impact. However, to date, little is known about the functions of lysine acetylation in this Phytopthora. Here, we conducted a lysine acetylome in P. sojae. Overall, 2197 lysine acetylation sites in 1150 proteins were identified. The modified proteins are involved in diverse biological processes and are localized to multiple cellular compartments. Importantly, 7 proteins involved in the pathogenicity or the secretion pathway of P. sojae were found to be acetylated. These data provide the first comprehensive view of the acetylome of P. sojae and serve as an important resource for functional analysis of lysine acetylation in plant pathogens. PMID:27412925

  5. Total chemically available (free and intrachain) lysine and furosine in pea, bean, and lentil sprouts.

    PubMed

    Rodríguez, Carmen; Frías, Juana; Vidal-Valverde, Concepción; Hernández, Amelia

    2007-12-12

    The effect of the germination of peas, beans, and lentils under differing conditions of illumination for different times on parameters linked to the Maillard reaction (chemically available free and intrachain lysine, lysine availability, and furosine) was evaluated. The chemically available free lysine content in the raw seeds of the three legumes was quite small compared to the chemically available intrachain lysine content, and furosine was detectable only in the beans and the lentils. The effect of germination was to increase lysine availability compared with levels in the raw seeds in all of the germinated samples, the smallest increase taking place in the lentils. In addition, furosine became detectable in all of the germinated samples. Quantities varied depending on the germination conditions but in all cases were higher than the quantities observed in the raw seeds. Linear correlations were observed to exist between some of the parameters considered in the three legumes tested. PMID:17973449

  6. Impact of dry heating on physicochemical properties of corn starch and lysine mixture.

    PubMed

    Ji, Ying; Yu, Jicheng; Xu, Yongbin; Zhang, Yinghui

    2016-10-01

    Corn starch was modified with lysine by dry heat treatment and to investigate how they can affect the pasting and structural properties of the treated starches. Dry heating with lysine reduced the pasting temperature and resulting in viscosity increase. The particle size of heated starch-lysine mixture increased, suggesting that starch granules were cross-linked to lysine. After dry heating, the onset temperature, peak temperature and conclusion temperature of corn starch-lysine mixture were lower than those of other starches. The degree of crystallinity decreased for the starch after dry heat treatment while these heated starch samples still have the same X-ray diffraction types as the original starch. PMID:27311503

  7. Structural Insights Into Amino Acid Binding and Gene Control by a Lysine Riboswitch

    SciTech Connect

    Serganov, A.; Huang, L; Patel, D

    2008-01-01

    In bacteria, the intracellular concentration of several amino acids is controlled by riboswitches1, 2, 3, 4. One of the important regulatory circuits involves lysine-specific riboswitches, which direct the biosynthesis and transport of lysine and precursors common for lysine and other amino acids. To understand the molecular basis of amino acid recognition by riboswitches, here we present the crystal structure of the 174-nucleotide sensing domain of the Thermotoga maritima lysine riboswitch in the lysine-bound (1.9 A) and free (3.1 A) states. The riboswitch features an unusual and intricate architecture, involving three-helical and two-helical bundles connected by a compact five-helical junction and stabilized by various long-range tertiary interactions. Lysine interacts with the junctional core of the riboswitch and is specifically recognized through shape-complementarity within the elongated binding pocket and through several direct and K+-mediated hydrogen bonds to its charged ends. Our structural and biochemical studies indicate preformation of the riboswitch scaffold and identify conformational changes associated with the formation of a stable lysine-bound state, which prevents alternative folding of the riboswitch and facilitates formation of downstream regulatory elements. We have also determined several structures of the riboswitch bound to different lysine analogues5, including antibiotics, in an effort to understand the ligand-binding capabilities of the lysine riboswitch and understand the nature of antibiotic resistance. Our results provide insights into a mechanism of lysine-riboswitch-dependent gene control at the molecular level, thereby contributing to continuing efforts at exploration of the pharmaceutical and biotechnological potential of riboswitches.

  8. Unbalance of L-lysine flux in Corynebacterium glutamicum and its use for the isolation of excretion-defective mutants.

    PubMed Central

    Vrljic, M; Kronemeyer, W; Sahm, H; Eggeling, L

    1995-01-01

    We found that the simple addition of L-methionine to the wild type of Corynebacterium glutamicum results in excretion of the cellular building block L-lysine up to rates of 2.5 nmol/min/mg (dry weight). Biochemical analyses revealed that L-methionine represses the homoserine dehydrogenase activity and reduces the intracellular L-threonine level from 7 to less than 2 mM. Since L-lysine synthesis is regulated mainly by L-threonine (plus L-lysine) availability, the result is enhanced flux towards L-lysine. This indicates a delicate and not well controlled type of flux control at the branch point of aspartate semialdehyde conversion to either L-lysine or L-threonine, probably due to the absence of isoenzymes in C. glutamicum. The inducible system of L-lysine excretion discovered was used to isolate mutants defective in the excretion of this amino acid. One such mutant characterized in detail accumulated 174 mM L-lysine in its cytosol without extracellular excretion of L-lysine, whereas the wild type accumulated 53 mM L-lysine in the cytosol and 5.9 mM L-lysine in the medium. The mutant was unaffected in L-lysine uptake or L-isoleucine or L-glutamate excretion, and also the membrane potential was unaltered. This mutant therefore represents a strain with a defect in an excretion system for the primary metabolite L-lysine. PMID:7608075

  9. Severe dietary lysine restriction affects growth and body composition and hepatic gene expression for nitrogen metabolism in growing rats.

    PubMed

    Kim, J; Lee, K S; Kwon, D-H; Bong, J J; Jeong, J Y; Nam, Y S; Lee, M S; Liu, X; Baik, M

    2014-02-01

    Dietary lysine restriction may differentially affect body growth and lipid and nitrogen metabolism, depending on the degree of lysine restriction. This study was conducted to examine the effect of dietary lysine restriction on growth and lipid and nitrogen metabolism with two different degree of lysine restriction. Isocaloric amino acid-defined diets containing 1.4% lysine (adequate), 0.70% lysine (50% moderate lysine restriction) and 0.35% lysine (75% severe lysine restriction) were fed from the age of 52 to 77 days for 25 days in male Sprague-Dawley rats. The 75% severe lysine restriction increased (p < 0.05) food intake, but retarded (p < 0.05) growth, increased (p < 0.05) liver and muscle lipid contents and abdominal fat accumulation, increased (p < 0.05) blood urea nitrogen levels and mRNA levels of the serine-synthesizing 3-phosphoglycerate dehydrogenase gene, but decreased (p < 0.05) urea cycle arginase gene mRNA levels. In contrast, the 50% lysine restriction did not significantly (p > 0.05) affect body growth and lipid and nitrogen metabolism. Our results demonstrate that severe 75% lysine restriction has detrimental effects on body growth and deregulate lipid and nitrogen metabolism. PMID:23441935

  10. Substrate specificity of the metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) assessed by mutagenesis and analysis of synthetic peptides: substrate residues distant from the scissile bond are critical for proteolysis.

    PubMed Central

    Laursen, Lisbeth S; Overgaard, Michael T; Nielsen, Claus G; Boldt, Henning B; Hopmann, Kathrin H; Conover, Cheryl A; Sottrup-Jensen, Lars; Giudice, Linda C; Oxvig, Claus

    2002-01-01

    Human pregnancy-associated plasma protein-A (PAPP-A) cleaves insulin-like growth factor (IGF) binding protein-4 (IGFBP-4), causing a dramatic reduction in its affinity for IGF-I and -II. Through this mechanism, PAPP-A is a regulator of IGF bioactivity in several systems, including the human ovary and the cardiovascular system. PAPP-A belongs to the metzincin superfamily of zinc metalloproteinases, and is the founding member of a fifth metzincin family, the pappalysins. Herein, we first determined that PAPP-A cleaves IGFBP-4 at a single site (Met-135/Lys-136), and we analysed the influence of ionic strength, pH and zinc ion concentration on the cleavage reaction. Secondly, we sought to delineate the role of substrate residues in PAPP-A-mediated cleavage by the construction and analysis of 30 IGFBP-4 mutants in which various residues were replaced by alanine, by the analysis of eight mutants of IGFBP-5 (found recently to be a second PAPP-A substrate), and by cleavage analysis of synthetic peptides derived from IGFBP-4. Our data reveal a complex mode of substrate recognition and/or binding, pointing at important roles for several basic residues located up to 16 residues N-terminal to the scissile bond. An unexpected parallel can be drawn with an intracellular enzyme, the mitochondrial processing peptidase, that may help us to understand properties of the pappalysins. Further, proteinase-resistant variants of IGFBP-4 and -5, presented here, will be useful tools for the study of proteolysis in cell-based systems, and our finding that a synthetic peptide can be cleaved by PAPP-A provides the basis for development of quantitative assays for the investigation of PAPP-A enzyme kinetics. PMID:12241545

  11. Coprecipitation of thermal lysine-rich proteinoids with polyribonucleotides

    NASA Technical Reports Server (NTRS)

    Lacey, J. C., Jr.; Yuki, A.; Fox, S. W.

    1979-01-01

    An experimental study was conducted to determine whether the precipitation of thermal proteinoids with homopolynucleotides can serve as a tool for studying the specificities between proteins and polynucleotides. Attention is given to exploring the best means of quantitation of the precipitate and the effect of varying the lysine content and the amount of Mg(2+) on the results. The formation of microparticles was monitored both by turbidity and by the mass of precipitate formed. Only under certain conditions was the turbidity a reliable indication of the amount of precipitate. Increasing concentration of Mg(2+) tended to displace proteinoid from the complex with polynucleotide. The results indicate that the interaction of thermal proteinoids with polynucleotides appears to be a suitable tool for studying specificities of interactions between proteins and nucleic acids.

  12. The oncometabolite 2-hydroxyglutarate inhibits histone lysine demethylases

    PubMed Central

    Chowdhury, Rasheduzzaman; Yeoh, Kar Kheng; Tian, Ya-Min; Hillringhaus, Lars; Bagg, Eleanor A; Rose, Nathan R; Leung, Ivanhoe K H; Li, Xuan S; Woon, Esther C Y; Yang, Ming; McDonough, Michael A; King, Oliver N; Clifton, Ian J; Klose, Robert J; Claridge, Timothy D W; Ratcliffe, Peter J; Schofield, Christopher J; Kawamura, Akane

    2011-01-01

    Mutations in isocitrate dehydrogenases (IDHs) have a gain-of-function effect leading to R(−)-2-hydroxyglutarate (R-2HG) accumulation. By using biochemical, structural and cellular assays, we show that either or both R- and S-2HG inhibit 2-oxoglutarate (2OG)-dependent oxygenases with varying potencies. Half-maximal inhibitory concentration (IC50) values for the R-form of 2HG varied from approximately 25 μM for the histone Nɛ-lysine demethylase JMJD2A to more than 5 mM for the hypoxia-inducible factor (HIF) prolyl hydroxylase. The results indicate that candidate oncogenic pathways in IDH-associated malignancy should include those that are regulated by other 2OG oxygenases than HIF hydroxylases, in particular those involving the regulation of histone methylation. PMID:21460794

  13. High-throughput screening to identify inhibitors of lysine demethylases

    PubMed Central

    Gale, Molly; Yan, Qin

    2015-01-01

    Lysine demethylases (KDMs) are epigenetic regulators whose dysfunction is implicated in the pathology of many human diseases including various types of cancer, inflammation and X-linked intellectual disability. Particular demethylases have been identified as promising therapeutic targets, and tremendous efforts are being devoted toward developing suitable small-molecule inhibitors for clinical and research use. Several high-throughput screening strategies have been developed to screen for small-molecule inhibitors of KDMs, each with advantages and disadvantages in terms of time, cost, effort, reliability and sensitivity. In this Special Report, we review and evaluate the high-throughput screening methods utilized for discovery of novel small-molecule KDM inhibitors. PMID:25687466

  14. Synthesis, Biodegradability, and Biocompatibility of Lysine Diisocyanate–Glucose Polymers

    PubMed Central

    ZHANG, JIAN-YING; BECKMAN, ERIC J.; HU, JING; YANG, GUO-GUANG; AGARWAL, SUDHA; HOLLINGER, JEFFREY O.

    2016-01-01

    The success of a tissue-engineering application depends on the use of suitable biomaterials that degrade in a timely manner and induce the least immunogenicity in the host. With this purpose in mind, we have attempted to synthesize a novel nontoxic biodegradable lysine diisocyanate (LDI)-and glucose-based polymer via polymerization of highly purified LDI with glucose and its subsequent hydration to form a spongy matrix. The LDI–glucose polymer was degradable in aqueous solutions at 37, 22, and 4°C, and yielded lysine and glucose as breakdown products. The degradation products of the LDI–glucose polymer did not significantly affect the pH of the solution. The physical properties of the polymer were found to be adequate for supporting cell growth in vitro, as evidenced by the fact that rabbit bone marrow stromal cells (BMSCs) attached to the polymer matrix, remained viable on its surface, and formed multilayered confluent cultures with retention of their phenotype over a period of 2 to 4 weeks. These observations suggest that the LDI–glucose polymer and its degradation products were nontoxic in vitro. Further examination in vivo over 8 weeks revealed that subcutaneous implantation of hydrated matrix degraded in vivo three times faster than in vitro. The implanted polymer was not immunogenic and did not induce antibody responses in the host. Histological analysis of the implanted polymer showed that LDI–glucose polymer induced a minimal foreign body reaction, with formation of a capsule around the degrading polymer. The results suggest that biodegradable peptide-based polymers can be synthesized, and may potentially find their way into biomedical applications because of their biodegradability and biocompatibility. PMID:12459056

  15. Autoactivation by a Candida glabrata copper metalloregulatory transcription factor requires critical minor groove interactions.

    PubMed Central

    Koch, K A; Thiele, D J

    1996-01-01

    Rapid transcriptional autoactivation of the Candida glabrata AMT1 copper metalloregulatory transcription factor gene is essential for survival in the presence of high extracellular copper concentrations. Analysis of the interactions between purified recombinant AMT1 protein and the AMT1 promoter metal regulatory element was carried out by a combination of missing-nucleoside analysis, ethylation interference, site-directed mutagenesis, and quantitative in vitro DNA binding studies. The results of these experiments demonstrate that monomeric AMT1 binds the metal regulatory element with very high affinity and utilizes critical contacts in both the major and minor grooves. A single adenosine residue in the minor groove, conserved in all known yeast Cu metalloregulatory transcription factor DNA binding sites, plays a critical role in both AMT1 DNA binding in vitro and Cu-responsive AMT1 gene transcription in vivo. Furthermore, a mutation in the AMT1 Cu-activated DNA binding domain which converts a single arginine, found in a conserved minor groove binding domain, to lysine markedly reduces AMT1 DNA binding affinity in vitro and results in a severe defect in the ability of C. glabrata cells to mount a protective response against Cu toxicity. PMID:8552101

  16. Acrolein sequestering ability of the endogenous tripeptide glycyl-histidyl-lysine (GHK): characterization of conjugation products by ESI-MSn and theoretical calculations.

    PubMed

    Beretta, Giangiacomo; Arlandini, Emanuele; Artali, Roberto; Anton, Josep M Garcia; Maffei Facino, R

    2008-07-15

    Acrolein (ACR) is a well-known carbonyl toxin produced by lipid peroxidation of polyunsaturated fatty acids, which is involved in several life-threatening pathologies such as Alzheimer disease, arteriosclerosis, diabetes, and nephropathy. The aim of this work was to study the quenching ability of the endogenous tripeptide glycyl-histidyl-lysine (GHK), a liver cell growth factor isolated from human plasma, towards the electrophilic aldehyde ACR and to characterize the reaction products by electrospray mass spectrometry (ESI-MS/MS infusion experiments; positive ion mode). The reaction of ACR (30 microM) with GHK (0.1, 0.25, 0.5, 1.0 mM) was followed by measuring aldehyde consumption by reverse-phase HPLC (phosphate buffer, pH 7.4); after 4h, when the aldehyde had completely disappeared; the reaction products were checked by ESI-MS/MS. Several products were detected in the GHK+ACR reaction (1:1). This indicates a complex reaction cascade involving the sequential addition of ACR (up to 3 mol) to the tripeptide GHK and, in particular, to the epsilon-amino group of the lysine residue and to the N(tau) and N(pi) of the histidine moiety. The Michael addition of two molecules of ACR to the epsilon-amino group of the lysine residue is followed by aldol condensation and dehydration to give the N-(3-formyl-3,4-dehydropiperidino) derivative. The results confirm that the ESI-MS/MS approach in a direct infusion experiment permits rapid profiling of the products of the GHK+ACR reaction. They firstly point to the potential medicinal use of GHK in the prevention of carbonyl stress-linked pathologies, and--second--help shed light on the physiological role of this histidine-containing tripeptide which is claimed to be an endogenous growth factor, but has never been shown to be an ACR quencher. PMID:18378108

  17. Genome-Wide Analysis of the Lysine Biosynthesis Pathway Network during Maize Seed Development

    PubMed Central

    Liu, Yuwei; Xie, Shaojun; Yu, Jingjuan

    2016-01-01

    Lysine is one of the most limiting essential amino acids for humans and livestock. The nutritional value of maize (Zea mays L.) is reduced by its poor lysine content. To better understand the lysine biosynthesis pathway in maize seed, we conducted a genome-wide analysis of the genes involved in lysine biosynthesis. We identified lysine biosynthesis pathway genes (LBPGs) and investigated whether a diaminopimelate pathway variant exists in maize. We analyzed two genes encoding the key enzyme dihydrodipicolinate synthase, and determined that they contribute differently to lysine synthesis during maize seed development. A coexpression network of LBPGs was constructed using RNA-sequencing data from 21 developmental stages of B73 maize seed. We found a large set of genes encoding ribosomal proteins, elongation factors and zein proteins that were coexpressed with LBPGs. The coexpressed genes were enriched in cellular metabolism terms and protein related terms. A phylogenetic analysis of the LBPGs from different plant species revealed different relationships. Additionally, six transcription factor (TF) families containing 13 TFs were identified as the Hub TFs of the LBPGs modules. Several expression quantitative trait loci of LBPGs were also identified. Our results should help to elucidate the lysine biosynthesis pathway network in maize seed. PMID:26829553

  18. Biofortification of rice with the essential amino acid lysine: molecular characterization, nutritional evaluation, and field performance.

    PubMed

    Yang, Qing-Qing; Zhang, Chang-Quan; Chan, Man-Ling; Zhao, Dong-Sheng; Chen, Jin-Zhu; Wang, Qing; Li, Qian-Feng; Yu, Heng-Xiu; Gu, Ming-Hong; Sun, Samuel Sai-Ming; Liu, Qiao-Quan

    2016-07-01

    Rice (Oryza sativa L.), a major staple crop worldwide, has limited levels of the essential amino acid lysine. We previously produced engineered rice with increased lysine content by expressing bacterial aspartate kinase and dihydrodipicolinate synthase and inhibiting rice lysine ketoglutarate reductase/saccharopine dehydrogenase activity. However, the grain quality, field performance, and integration patterns of the transgenes in these lysine-enriched lines remain unclear. In the present study, we selected several elite transgenic lines with endosperm-specific or constitutive regulation of the above key enzymes but lacking the selectable marker gene. All target transgenes were integrated into the intragenic region in the rice genome. Two pyramid transgenic lines (High Free Lysine; HFL1 and HFL2) with free lysine levels in seeds up to 25-fold that of wild type were obtained via a combination of the above two transgenic events. We observed a dramatic increase in total free amino acids and a slight increase in total protein content in both pyramid lines. Moreover, the general physicochemical properties were improved in pyramid transgenic rice, but the starch composition was not affected. Field trials indicated that the growth of HFL transgenic rice was normal, except for a slight difference in plant height and grain colour. Taken together, these findings will be useful for the potential commercialization of high-lysine transgenic rice. PMID:27252467

  19. Voluntary wheel running is beneficial to the amino acid profile of lysine-deficient rats.

    PubMed

    Nagao, Kenji; Bannai, Makoto; Seki, Shinobu; Kawai, Nobuhiro; Mori, Masato; Takahashi, Michio

    2010-06-01

    Rats voluntarily run up to a dozen kilometers per night when their cages are equipped with a running wheel. Daily voluntary running is generally thought to enhance protein turnover. Thus, we sought to determine whether running worsens or improves protein degradation caused by a lysine-deficient diet and whether it changes the utilization of free amino acids released by proteolysis. Rats were fed a lysine-deficient diet and were given free access to a running wheel or remained sedentary (control) for 4 wk. Amino acid levels in plasma, muscle, and liver were measured together with plasma insulin levels and tissue weight. The lysine-deficient diet induced anorexia, skeletal muscle loss, and serine and threonine aminoacidemia, and it depleted plasma insulin and essential amino acids in skeletal muscle. Allowing rats to run voluntarily improved these symptoms; thus, voluntary wheel running made the rats less susceptible to dietary lysine deficiency. Amelioration of the declines in muscular leucine and plasma insulin observed in running rats could contribute to protein synthesis together with the enhanced availability of lysine and other essential amino acids in skeletal muscle. These results indicate that voluntary wheel running under lysine-deficient conditions does not enhance protein catabolism; on the contrary, it accelerates protein synthesis and contributes to the maintenance of muscle mass. The intense nocturnal voluntary running that characterizes rodents might be an adaptation of lysine-deficient grain eaters that allows them to maximize opportunities for food acquisition. PMID:20233939

  20. Acetylation of lysine 109 modulates pregnane X receptor DNA binding and tr