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PubMed1992

DNA base modifications induced in isolated human chromatin by NADH dehydrogenase-catalyzed reduction of doxorubicin.

The antineoplastic benzanthroquinone drug doxorubicin can undergo flavoenzyme-catalyzed one-electron reduction which, in an aerobic environment, leads to the generation of oxygen-derived species. We therefore sought to determine whether doxorubicin in the presence of NADH dehydrogenase and the transition metal ions Fe(III) or Cu(II) induces DNA base modifications in isolated human chromatin. NADH dehydrogenase-catalyzed reduction of doxorubicin (25-100 microM) caused hydroxyl radical production detected as methane generated from dimethyl sulfoxide; addition of isolated human chromatin to the system produced a concentration-dependent quenching of detectable hydroxyl radical formation. Doxorubicin (5-50 microM)-stimulated enzyme-catalyzed oxidation of NADH was also diminished, but still detectable, in the presence of chromatin. Doxorubicin-induced DNA base modifications in chromatin were measured by gas chromatography/mass spectrometry with selected-ion monitoring. Production of modified bases required the addition of transition metal ion and was enhanced by the addition of active flavoenzyme. The non-redox cycling analogue 5-iminodaunorubicin induced significantly less base modification than did doxorubicin. In the presence of Fe(III), NADH dehydrogenase-catalyzed reduction of doxorubicin caused enhancement in the content of all modified bases over control levels. Substitution of Cu(II) for Fe(III) altered both the degree and the pattern of doxorubicin/NADH dehydrogenase-induced base modifications. The scavengers of hydroxyl radical mannitol and dimethyl sulfoxide or catalase did not significantly affect doxorubicin/NADH/NADH dehydrogenase/transition metal ion-induced base modifications. Superoxide dismutase further enhanced production of all base modifications. The data demonstrate that flavoenzyme-catalyzed redox cycling of doxorubicin generates typical hydroxyl radical-induced base modifications in the DNA of isolated human chromatin, suggesting a possible mechanism for the mutagenicity of doxorubicin in vivo.

Chromatin↗
PubMed1992

Structural properties of arrestin studied by chemical modification and circular dichroism.

A unique conformation of arrestin is crucial for its interaction with phosphorylated photolyzed rhodopsin. Conformational changes in arrestin were investigated using chemical modification and circular dichroism. We studied the kinetics of sulfhydryl modification of bovine arrestin in order to determine whether its conformation is altered by the presence of ligands or salts at different ionic strengths. We found that all three cysteines (stoichiometry was 2.7 +/- 0.06 3-carboxy-4-nitrophenyl sulfide (NbS)/arrestin) are accessible for modification by NbS2. Under pseudo-first-order conditions (30-100-fold excess of NbS2 over arrestin), the modifications of the 3 cysteines are indistinguishable. At higher concentrations of NbS2 (150-300-fold excess), the pseudo-first-order plot is not linear, and the reaction can be resolved into two processes that involve two classes of sulfhydryl groups. Addition of CaCl2, MgCl2, inorganic phosphate, MgATP, or MgGTP had little effect on the rate of modification of the cysteine residues; however, heparin and inositol hexakisphosphate, which have been shown to induce conformational changes in arrestin, block modification of one sulfhydryl group of arrestin and accelerate the modification of the remaining two. Analysis of CD spectra revealed that arrestin has virtually no alpha-helical structure, about 40% beta-structure, about 18% beta-turns, and about 40% other structure. The CD spectrum for arrestin did not change in the presence of heparin. These studies suggest that arrestin exists in equilibrium between two or more conformational states. However, it is proposed that conversion between these conformations occur without altering significantly the secondary structure of arrestin.

Adenosine Triphosphate↗
PubMed1994

Intracellular early and late modifications of human apolipoprotein A-II. Effect of glutamine- +1 to leucine substitution.

It has been shown previously that apoA-II undergoes several intracellular modifications in HepG2 cells (Hussain & Zannis, 1990). In the present study, we have generated permanent cell lines in mouse C127 cells which express the normal apoA-II gene and a mutated form in which Gln+1 was substituted with Leu (Leu+1). This modification was designed to prevent cyclization of the N-terminal glutamine of apoA-II and thus identify the isoproteins which are precursors and products of the N-terminal cyclization reaction. The C127-expression cells were also utilized to study the cellular compartments where the apoA-II modifications occur as well as the importance of the modifications for apoA-II trafficking and secretion. We have found that apoA-II (Gln+1) synthesized by C127 and HepG2 cells had similar isoproteins. In both cell types, unmodified pro-apoA-II, designated isoprotein 3, had a similar isoelectric point as the cell-free translation product of apoA-II mRNA, suggesting that isoprotein 3 results from cleavage of the signal peptide. Isoprotein 3 represents an unmodified apoA-II isoprotein and undergoes an early modification into a more acidic isoprotein 1, which differs from isoprotein 3 by two negative charges. Brefeldin A treatment of the cells did not prevent the formation of isoprotein 1, suggesting that this modification occurs in a pre-Golgi compartment. Neuraminidase treatment of secreted apoA-II isoproteins did not affect isoprotein 1, indicating that it is not sialylated isoprotein. Isoprotein 1 undergoes further modifications which are consistent with cleavage of the propeptide, N-terminal cyclization and sialylation most likely resulting from O-glycosylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoprotein A-II↗
PubMed1996

Essential cysteines in 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase from Escherichia coli: analysis by chemical modification and site-directed mutagenesis.

The enzyme 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase (EC 4.1. 2.16) (KDO 8-P synthase) that catalyzes the condensation of D-arabinose 5-phosphate (A 5-P) with phosphoenolpyruvate (PEP) to give 3-deoxy-D-manno-octulosonic acid 8-phosphate (KDO 8-P) and inorganic phosphate (Pi) was inactivated by the thiol-modifying reagents 5,5-dithiobis (2-nitrobenzoate) (DTNB) and methyl methanethiosulfonate (MMTS). Reaction of cloned native KDO 8-P synthase with DTNB correlated with modification of two of the four cysteine sulfhydryls per monomer of enzyme and total loss of enzymatic activity which could be partially restored by treatment with dithiothreitol (DTT). Cyanolysis of the DTNB-inactivated enzyme with KCN led to the elimination of 2 equiv of 5-thio-2-nitrobenzoate and partial recovery of activity. The presence of either substrate(s) or product(s) provided no protection against inactivation nor affected the number of cysteines modified, indicating that the cysteines modified are most likely not at the active site of KDO 8-P synthase. Titration of denatured enzyme with DTNB resulted in the modification of all four cysteines. After treatment of native enzyme with MMTS, no cysteines could be titrated with DTNB and no enzymatic activity could be detected. Treatment of the MMTS-inactivated KDO 8-P synthase with DTT resulted in restoration of enzymatic activity and the presence of two DTNB-titratable cysteine residues. Based on these observations and a report that KDO 8-P synthase is inactivated in a time-dependent manner with 3-bromopyruvate and that the substrate PEP protects against this inactivation, all four cysteines (38, 166, 206, and 249) were individually mutated to alanines via a modified PCR methodology. The C206A and C249A mutants were both enzymatically active with K(m) and Vmax values approximately identical to those of wild-type KDO 8-P synthase, and both native mutants reacted with DTNB to modify only one of the three remaining cysteine sulfhydryls per monomer of enzyme. Titration of denatured C206A and C249A mutants resulted in the modification of three cysteines. The C38A and C166A mutants were both for the most part enzymatically inactive. Titration of native C38A and C166A with DTNB resulted in modification of two cysteines while titration of the denatured mutant protein resulted in modification of the three remaining cysteines. Circular dichroism measurements of wild-type KDO 8-P synthase and the four C --> A mutants indicate modest but significant changes in the structure of the mutants. These results indicate that C206 and C249 in native KDO 8-P synthase are readily accessible to the modification reagent DTNB and therefore inactivation may result from structural changes in the DTNB-modified KDO 8-P synthase or blockage of access of substrates to the active site. The C38 and C166 in native KDO 8-P synthase are inaccessible to the modification reagent DTNB, indicating that they are located in the interior of KDO 8-P synthase, and loss of activity in the C38A and C166A mutants suggests their essentiality in the KDO 8-P synthase reaction.

Aldehyde-Lyases↗
PubMed1999

Peroxynitrite modification of protein thiols: oxidation, nitrosylation, and S-glutathiolation of functionally important cysteine residue(s) in the sarcoplasmic reticulum Ca-ATPase.

Skeletal muscle contraction and relaxation is efficiently modulated through the reaction of reactive oxygen-nitrogen species with sarcoplasmic reticulum protein thiols in vivo. However, the exact locations of functionally important modifications are at present unknown. Here, we determine by HPLC-MS that the modification of one (out of 24) Cys residue of the sarcoplasmic reticulum (SR) Ca-ATPase isoform SERCA1, Cys(349), by peroxynitrite is sufficient for the modulation of enzyme activity. Despite the size and nature of the SR Ca-ATPase, a 110 kDa membrane protein, identification and quantitation of Cys modification was achieved through labeling with 4-(dimethylamino)phenylazophenyl-4'-maleimide (DABMI) and/or N-(2-iodoethyl)trifluoroacetamide (IE-TFA) followed by an exhaustive tryptic digestion and on-line HPLC-UV-electrospray MS analysis. The reaction with IE-TFA generates aminoethylcysteine, a new trypsin cleavage site, which allows the production of specific peptide fragments that are diagnostic for IE-TFA labeling, conveniently identified by mass spectrometry. Exposure of the SR Ca-ATPase to low concentrations (0.1 mM) of peroxynitrite resulted in the fully reversible chemical modification of Cys at positions 344, 349, 471, 498, 525, and 614 (nitrosylation of Cys(344) and Cys(349) was seen), whereas higher concentrations of peroxynitrite (0.45 mM) additionally affected Cys residues at positions 636, 670, and 674. When the SR Ca-ATPase was exposed to 0.45 mM peroxynitrite in the presence of 5.0 mM glutathione (GSH), thiol modification became partially reversible and S-glutathiolation was detected for Cys residues at positions 344, 349, 364, 498, 525, and 614. The extent of enzyme inactivation (determined previously) quantitatively correlated with the loss of labeling efficiency (i) of a single Cys residue and (ii) of the tryptic fragment containing both Cys(344) and Cys(349). Earlier results had shown that the independent selective modification of Cys(344) is functionally insignificant [Kawakita, M., and Yamashita, T. (1987) J. Biochem. (Tokyo) 102, 103-109]. Thus, we conclude that modification of only Cys(349) is responsible for the modulation of the SR Ca-ATPase activity by peroxynitrite.

Acetamides↗
PubMed2003

Binary switches and modification cassettes in histone biology and beyond.

An immense number of post-translational modifications on histone proteins have been described and additional sites of modification are still being uncovered. Whereas many direct and indirect connections between certain histone modifications and distinct biological phenomena have now been established, concepts for comprehending the extreme density and variety of these covalent modifications are lacking. Here, we formally introduce localized 'binary switches' and 'modification cassettes' as new concepts in histone biology, elucidating mechanisms that might govern the biological readout of distinct modification patterns. Specifically, our hypotheses provide missing models for the dynamic readout of stable histone modifications and offer explanations for several long-standing questions embedded in the literature. Our ideas might also apply to non-histone proteins and are open to direct experimental examination.

Gene Expression Regulation↗
PubMed2005

Global histone modification patterns predict risk of prostate cancer recurrence.

Aberrations in post-translational modifications of histones have been shown to occur in cancer cells but only at individual promoters; they have not been related to clinical outcome. Other than being targeted to promoters, modifications of histones, such as acetylation and methylation of lysine and arginine residues, also occur over large regions of chromatin including coding regions and non-promoter sequences, which are referred to as global histone modifications. Here we show that changes in global levels of individual histone modifications are also associated with cancer and that these changes are predictive of clinical outcome. Through immunohistochemical staining of primary prostatectomy tissue samples, we determined the percentage of cells that stained for the histone acetylation and dimethylation of five residues in histones H3 and H4. Grouping of samples with similar patterns of modifications identified two disease subtypes with distinct risks of tumour recurrence in patients with low-grade prostate cancer. These histone modification patterns were predictors of outcome independently of tumour stage, preoperative prostate-specific antigen levels, and capsule invasion. Thus, widespread changes in specific histone modifications indicate previously undescribed molecular heterogeneity in prostate cancer and might underlie the broad range of clinical behaviour in cancer patients.

Acetylation↗
PubMed1987

Chemical modification studies on a lectin from Saccharomyces cerevisiae (baker's yeast).

The effect of chemical modification on a galactose-specific lectin isolated from a fatty acid auxotroph of Saccharomyces cerevisiae was investigated in order to identify the type of amino acids involved in its agglutinating activity. Modification of 50 free amino groups with succinic anhydride or citraconic anhydride led to an almost complete loss of activity. This could not be protected by the inhibitory sugar methyl alpha-D-galactopyranoside. Treatment with N-bromosuccinimide and N-acetylimidazole, for the modification of tryptophan and tyrosine residues, did not affect lectin activity. Modification of carboxy groups with glycine ethyl ester greatly affected lectin activity, although sugars afford partial protection. Modification of four thiol groups with N-ethylmaleimide was accompanied by a loss of 85% of the agglutinating activity, and two thiol groups were found to be present at the sugar-binding site of the lectin. Modification of 18 arginine residues with cyclohexane-1,2-dione and 26 histidine residues with ethoxyformic anhydride led to a loss of lectin activity. However, in these cases, modification was not protected by the abovementioned inhibitory sugar, suggesting the absence of these groups at the sugar-binding site. In all the cases, immunodiffusion studies with modified lectin showed no gross structural changes which could disrupt antigenic sites of the lectin.

Amino Acids↗
PubMed1994

Diethyl pyrocarbonate modification of the ryanodine receptor/Ca2+ channel from skeletal muscle.

Exposure of junctional sarcoplasmic reticulum (SR) membranes or purified ryanodine receptor to the histidine-specific reagent diethyl pyrocarbonate (DEPC) led to concentration- and time-dependent inactivation of ryanodine binding. The pH-dependence of the inactivation of ryanodine binding by DEPC and the reversal of this inactivation by hydroxylamine suggests the modification of histidine residue(s) by the reagent. Kinetic analysis of the time course of inactivation of ryanodine binding by DEPC suggests that the inactivation resulted from modification of a single class of histidine residue per ryanodine-binding site. The degree of inactivation of ryanodine binding by DEPC was decreased when high NaCl concentrations were present in the modification medium. The binding affinities for ryanodine and Ca2+ were not altered by DEPC modification. This modification decreased the total ryanodine-binding sites. DEPC modification increased the Ca(2+)-permeability of the SR vesicles. A variety of bivalent cations prevented the DEPC inactivation of ryanodine binding in a series of decreasing efficiency: Mn2+ > Ba2+ > Mg2+ > Ca2+, similar to their effectiveness in inhibiting ryanodine binding. It is suggested that a histidine residue(s) in the ryanodine receptor is involved, either in the binding of Ca2+, or in a conformational change that may be required for Ca2+ binding to its binding site(s). This modification of the ryanodine receptor resulted in inactivation of ryanodine binding and activation of Ca2+ release.

Animals↗
PubMed1994

Chemical modification of aminopeptidase isolated from Pronase.

Chemical modification of aminopeptidase from pronase has revealed two important histidines in enzyme catalysis. In the absence of metal ions, modification of the readily-modified histidine (pKa 6.9 +/- 0.5) results in a drastic loss of activity, indicating that this residue is indispensible for enzyme activity. In the presence of CaCl2, the modified enzyme still retains approx. 60% of the activity, whereas modification of another histidine (pKa 7.7 +/- 0.2) leads to a dramatic loss of activity. In fact, the enzyme with the first histidine being modified is active only in the presence of metal ions. Moreover, modification of the second histidine is prevented by the presence of Ca(II). These results indicate that the second histidine is serving as a ligand for Ca(II) and the bound Ca(II) is directly involved in enzyme catalysis. The c.d. spectra of the modified and unmodified enzymes in the absence or presence of CaCl2 are all very similar, indicating that no gross conformational changes in protein occur upon modification or by the presence of Ca(II). Modification of both histidines is prevented by the presence of a competitive inhibitor, suggesting that they are located in the active centre. Modification of 11 amino groups, two tyrosines, or four arginines causes no appreciable inactivation of the enzyme, indicating that these residues are not directly involved in enzyme catalysis.

Aminopeptidases↗
PubMed2003

In vitro and in vivo lipolysis of plasma triglycerides increases the resistance to oxidative modification of low-density lipoproteins.

BACKGROUND: The majority of studies on low-density lipoprotein (LDL) particle size and susceptibility to oxidative modification have been either descriptive or interventional, but there are few mechanistic studies. MATERIALS AND METHODS: Effects of exhaustive in vitro and in vivo lipolysis of serum and plasma triglycerides, respectively, by lipoprotein lipase (LPL) were investigated in healthy normotriglyceridemic men. The LDL end-product of lipolysis of very low-density lipoprotein (VLDL) underwent compositional analysis, gradient gel electrophoresis and an assessment of resistance to copper-induced oxidative modification. RESULTS: The LDL particle contents of free fatty acid and alpha-tocopherol increased, whereas the contents of free and esterified cholesterol, alpha-carotene and coenzyme Q10 decreased upon incubation of serum with LPL in vitro. The LDL particle size decreased and the resistance to the oxidative modification of LDL increased. Lipolysis of plasma triglycerides in vivo, achieved by intravenous injection of heparin, did not alter the LDL particle size but increased the resistance to the oxidative modification of LDL. This change was accompanied by an increase in the LDL particle content of alpha-tocopherol, whereas the free fatty acid content was unaltered. CONCLUSIONS: The results show that the increased resistance to oxidative modification of LDL after lipolysis of plasma triglycerides was concomitant with an increased LDL particle content of alpha-tocopherol, and that free fatty acids did not seem to contribute to the increased resistance to oxidative modification of LDL in vivo. Furthermore, our data indicate that the resistance of LDL to oxidative modification is not dependent on particle size.

Aged↗
PubMed2001

Behaviour modification and gentle teaching workshops: management of children with learning disabilities exhibiting challenging behaviour and implications for learning disability nursing.

BACKGROUND: Challenging behaviours (behaviour difficulties) represent a problem of considerable clinical significance for learning disability nurses, and a source of much human distress. Gentle teaching is a relatively new approach to dealing with behavioural difficulties, and has been received with enthusiasm by clinicians, but has so far received little empirical support. The current study attempted to compare gentle teaching with a well-established alternative (behaviour modification) and a control group. OBJECTIVES: To examine the comparative effectiveness of gentle teaching, behaviour modification and control interventions for challenging behaviour amongst children with learning disabilities. DESIGN: Nonrandomized controlled trial. SETTING: Service users' homes in East Yorkshire. PARTICIPANTS: Seventy-seven children who presented with learning disabilities and challenging behaviour (behaviour difficulties) and their parents. PROCEDURE: One-day workshops in were offered by recognized authorities in either behaviour modification or gentle teaching that were not otherwise involved with the research project. Forty-one participants were recruited to the gentle teaching condition; 36 to behaviour modification; 26 to the control group. Random allocation was not possible, because of the slow uptake by interested parents. Measures was preintervention, and at assessment points up until 12 months following intervention. ANALYSIS: Quantitative analysis of pre-post differences between the groups, using t-test. RESULTS: In general, no significant differences were found between the treatment groups and controls. Significant improvements were found for both gentle teachingand behaviour modification children over controls on the AAMR ABS XVII (social engagement) subscale. Controls had more contact with medical practitioner (GP) services than behaviour modification children and less than gentle teaching children. CONCLUSION: Although very few differences were found between the three groups, those that did exist generally favoured behaviour modification. Implications for service provision and learning disability nursing practice are described.

Adolescent↗
PubMed2003

Age-related modifications of the morphological organization of pituicytes are associated with alteration of the GABAergic and dopaminergic innervation afferent to the neurohypophysial lobe.

Ageing is known to induce a marked activation of astrocytes within various regions of the central nervous system. To date, the age-related factors responsible for these modifications are unknown. The neural lobe of the hypophysis (NL) is a particular brain region which does not contain neurons but does contain specialized astrocytes, called pituicytes, and numerous terminals of afferent axons, including (i) peptidergic neurohypophysial axons which terminate on the NL blood vessels, and (ii) axons containing both gamma amino-butyric acid (GABA) and dopamine (DA) which form contacts with pituicytes. Because evidence has recently been provided that GABA signalling mediates the morphological organization of astrocytes, the present study was designed to determine whether modifications of pituicytes during ageing were associated with modifications of the GABAergic axons innervating the NL. We show here that, in adult rats, GABA/DA axons form preferential synaptic-like contacts with pituicytes which express both GABAA and D2 dopamine receptors. We then show that, during ageing, pituicytes undergo dramatic modifications of their morphology, correlatively with marked modifications of the GABA/DA fibres innervating the NL. Lastly, in vitro experiments indicate that modifications of the morphology of pituicytes similar to those observed during ageing were obtained by incubating isolated NL of adult rats with a GABAA receptor agonist and/or a D2 dopamine receptor antagonist, whereas inverse modifications were observed when NL of aged rats were incubated with a GABAA receptor antagonist and a D2 dopamine receptor agonist. Taken together, these data suggest that the age-related morphological changes of pituicytes result from the alteration of the GABA/DAergic innervation of the NL.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed2004

Histone H3.3 is enriched in covalent modifications associated with active chromatin.

Chromatin states can be distinguished by differential covalent modifications of histones or by utilization of histone variants. Chromatin associated with transcriptionally active loci becomes enriched for histones with particular lysine modifications and accumulates the H3.3 histone variant, the substrate for replication-independent nucleosome assembly. However, studies of modifications at particular loci have not distinguished between histone variants, so the relationship among modifications, histone variants, and nucleosome assembly pathways is unclear. To address this uncertainty, we have quantified the relative abundance of H3 and H3.3 and their lysine modifications. Using a Drosophila cell line system in which H3.3 has been shown to specifically package active loci, we found that H3.3 accounts for approximately 25% of total histone 3 in bulk chromatin, enough to package essentially all actively transcribed genes. MS and antibody characterization of separated histone 3 fractions revealed that H3.3 is relatively enriched in modifications associated with transcriptional activity and deficient in dimethyl lysine-9, which is abundant in heterochromatin. To explain enrichment on alternative variants, we propose that histone modifications are tied to the alternative nucleosome assembly pathways that use primarily H3 at replication forks and H3.3 at actively transcribed genes in a replication-independent manner.

Animals↗
PubMed2004

Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure.

A dynamic programming algorithm for prediction of RNA secondary structure has been revised to accommodate folding constraints determined by chemical modification and to include free energy increments for coaxial stacking of helices when they are either adjacent or separated by a single mismatch. Furthermore, free energy parameters are revised to account for recent experimental results for terminal mismatches and hairpin, bulge, internal, and multibranch loops. To demonstrate the applicability of this method, in vivo modification was performed on 5S rRNA in both Escherichia coli and Candida albicans with 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate, dimethyl sulfate, and kethoxal. The percentage of known base pairs in the predicted structure increased from 26.3% to 86.8% for the E. coli sequence by using modification constraints. For C. albicans, the accuracy remained 87.5% both with and without modification data. On average, for these sequences and a set of 14 sequences with known secondary structure and chemical modification data taken from the literature, accuracy improves from 67% to 76%. This enhancement primarily reflects improvement for three sequences that are predicted with <40% accuracy on the basis of energetics alone. For these sequences, inclusion of chemical modification constraints improves the average accuracy from 28% to 78%. For the 11 sequences with <6% pseudoknotted base pairs, structures predicted with constraints from chemical modification contain on average 84% of known canonical base pairs.

Algorithms↗
PubMed2000

2'-O,4'-C-methylene bridged nucleic acid modification promotes pyrimidine motif triplex DNA formation at physiological pH: thermodynamic and kinetic studies.

Extreme instability of pyrimidine motif triplex DNA at physiological pH severely limits its use in an artificial control of gene expression in vivo. Stabilization of the pyrimidine motif triplex at physiological pH is, therefore, crucial in improving its therapeutic potential. To this end, we have investigated the thermodynamic and kinetic effects of our previously reported chemical modification, 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) modification of triplex-forming oligonucleotide (TFO), on pyrimidine motif triplex formation at physiological pH. The thermodynamic analyses indicated that the 2',4'-BNA modification of TFO increased the binding constant of the pyrimidine motif triplex formation at neutral pH by approximately 20 times. The number and position of the 2',4'-BNA modification introduced into the TFO did not significantly affect the magnitude of the increase in the binding constant. The consideration of the observed thermodynamic parameters suggested that the increased rigidity itself of the 2',4'-BNA-modified TFO in the free state relative to the unmodified TFO may enable the significant increase in the binding constant at neutral pH. Kinetic data demonstrated that the observed increase in the binding constant at neutral pH by the 2',4'-BNA modification of TFO resulted from the considerable decrease in the dissociation rate constant. Our results certainly support the idea that the 2',4'-BNA modification of TFO could be a key chemical modification and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.

DNA↗
PubMed2005

Mapping post-translational modifications of the histone variant MacroH2A1 using tandem mass spectrometry.

Post-translational histone modifications modulate chromatin-templated processes and therefore affect cellular proliferation, growth, and development. Although post-translational modifications on the core histones have been under intense investigation for several years, the modifications on variant histones are poorly understood. We used tandem mass spectrometry to identify covalent modifications on a histone H2A variant, macroH2A1.2. MacroH2A1.2 can be monoubiquitinated; however, the site of monoubiquitination has not been documented. In this study we used green fluorescent protein-tagged macroH2A1.2 to determine that Lys(115) is a site of ubiquitination. In addition, we found that this variant H2A is methylated on the epsilon amino group of lysine residues Lys(17), Lys(122), and Lys(238) and phosphorylated on Thr(128). Three of these modifications were also found to be present in the endogenous protein by mass spectrometric analysis. These results provide the first direct evidence that multiple post-translational modifications are imposed on macroH2A1.2, suggesting that, like canonical H2A, this variant H2A is subject to regulation by combinatorial use of covalent modifications.

Histones↗
PubMed2005

Identification of the major site of O-linked beta-N-acetylglucosamine modification in the C terminus of insulin receptor substrate-1.

Signal transduction from the insulin receptor to downstream effectors is attenuated by phosphorylation at a number of Ser/Thr residues of insulin receptor substrate-1 (IRS-1) resulting in resistance to insulin action, the hallmark of type II diabetes. Ser/Thr residues can also be reversibly glycosylated by O-linked beta-N-acetylglucosamine (O-GlcNAc) monosaccharide, a dynamic posttranslational modification that offers an alternative means of protein regulation to phosphorylation. To identify sites of O-GlcNAc modification in IRS-1, recombinant rat IRS-1 isolated from HEK293 cells was analyzed by two complementary mass spectrometric methods. Using data-dependent neutral loss MS3 mass spectrometry, MS/MS data were scanned for peptides that exhibited a neutral loss corresponding to the mass of N-acetylglucosamine upon dissociation in an ion trap. This methodology provided sequence coverage of 84% of the protein, permitted identification of a novel site of phosphorylation at Thr-1045, and facilitated the detection of an O-GlcNAc-modified peptide of IRS-1 at residues 1027-1073. The level of O-GlcNAc modification of this peptide increased when cells were grown under conditions of high glucose with or without chronic insulin stimulation or in the presence of an inhibitor of the O-GlcNAcase enzyme. To map the exact site of O-GlcNAc modification, IRS-1 peptides were chemically derivatized with dithiothreitol following beta-elimination and Michael addition prior to LC-MS/MS. This approach revealed Ser-1036 as the site of O-GlcNAc modification. Site-directed mutagenesis and Western blotting with an anti-O-GlcNAc antibody suggested that Ser-1036 is the major site of O-GlcNAc modification of IRS-1. Identification of this site will facilitate exploring the biological significance of the O-GlcNAc modification.

Acetylglucosamine↗