Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “modification”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,135 records · Page 63Linked to original sources
PubMed2005

Specific correlation between the wobble modification deficiency in mutant tRNAs and the clinical features of a human mitochondrial disease.

Mutations in mtDNA are responsible for a variety of mitochondrial diseases, where the mitochondrial tRNA(Leu(UUR)) gene has especially hot spots for pathogenic mutations. Clinical features often depend on the tRNA species and/or positions of the mutations; however, molecular pathogenesis elucidating the relation between the location of the mutations and their leading phenotype are not fully understood. We report here that mitochondrial tRNAs(Leu(UUR)) harboring one of five mutations found in tissues from patients with symptoms of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) (A3243G, G3244A, T3258C, T3271C, and T3291C) lacked the normal taurine-containing modification (5-taurinomethyluridine) at the anticodon wobble position. In contrast, mitochondrial tRNAs(Leu(UUR)) with different mutations found in patients that have mitochondrial diseases but do not show the MELAS symptoms (G3242A, T3250C, C3254T, and A3280G) had the normal 5-taurinomethyluridine modifications. These observations were made by using a modified primer extension technique that can detect the modification deficiency in the extremely limited quantities of mutant tRNAs obtainable from patient tissues. These results strongly suggest deficient wobble modification could be a key molecular factor responsible for the phenotypic features of MELAS, which can explain why the different MELAS-associated mutations result in indistinguishable clinical features.

Anticodon↗
PubMed2006

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

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 N(6)-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 N(6)-amino groups of lysine side chains. A liquid chromatography (LC)-tandem mass spectrometry (MS) method was developed to quantify the resulting N(6)-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 N(6)-formyl-lysine, with this nucleosome linker-selective DNA-cleaving agent causing selective N(6)-formylation of the linker histone H1. The N(6)-formyl-lysine residue appears to represent an endogenous histone secondary modification, one that bears chemical similarity to lysine N(6)-acetylation recognized as an important determinant of gene expression in mammalian cells. The N(6)-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.

Acetylation↗
PubMed1979

Paracatalytic modification of aldolase: a side reaction of the catalytic cycle resulting in irreversible blocking of two active-site lysyl residues.

Paracatalytic enzyme modifications result from the oxidation of enzyme-substrate carbanions by extrinsic oxidants. During the oxidation of enzyme-activated substrates, transiently reactive intermediates are generated which, without being released from the enzyme, modify groups at the active site. For enzymes producing carbanion intermediates, the combination of the normal substrate with a suitable electron acceptor has thus been proposed as a highly specific binary system for their active site-directed modification. In this study, the structural features of paracatalytically modified fructose-1,6-bisphosphate aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13) from rabbit muscle have been elucidated. This enzyme is completely inactivated within 60 min in the presence of fructose 1,6-bisphosphate in saturating concentration and 0.5 mM hexacyanoferrate(III) (pH 7.6, 25 degrees C). The inactivation is caused by covalent incorporation of one triosephosphate derivative per subunit. Peptide analysis showed that the triosephosphate derivative forms an intrachain crosslink between lysine-146 and lysine-227. According to previous independent experimental evidence, both lysyl residues are located at the active site: the epsilon-amino group of lysine-227 forms a Schiff base intermediate with the carbonyl group of the substrate [Lai, C. Y., Nakai, N. & Chang, D. (1974) Science 183, 1204-1206] and alkylation of lysine-146 by the affinity labeling reagent N-bromoacetylethanolamine phosphate inactivates the enzyme [Hartman, F. C. & Brown, J. P. (1976) J. Biol. Chem. 251, 3057-3062]. The present data thus establish paracatalytic modification as a mode of active site-directed enzyme modification.

Animals↗
PubMed1981

Delivery of hexosaminidase A to the cerebrum after osmotic modification of the blood--brain barrier.

The present studies were undertaken to evaluate the possibility that hexosaminidase A, the enzyme deficient in Tay--Sachs disease, could be effectively delivered to brain. Previous studies from our laboratory have shown that hypertonic mannitol can be used to osmotically produce reversible disruption of the blood--brain barrier in animals (rat and dog) and man without significant neurotoxicity and that such barrier modification significantly increases the delivery of cytoreductive chemotherapy agents to selected areas of brain. By using the rat model of blood--brain barrier modification and radiolabeled enzyme, increased hexosaminidase A delivery to brain has been demonstrated in more than 85 animals. The time of injection of hexosaminidase A after blood--brain barrier disruption is critical for maximum delivery. Rapid (over 30 sec) intra-arterial administration of hexosaminidase A immediately after blood--brain barrier disruption resulted in a marked increase in enzyme delivery to the brain when compared with controls without prior barrier disruption. When the enzyme was administered 15-20 min after barrier disruption, approximately 50% less hexosaminidase A was delivered; when given 60-120 min after barrier modification, the amount delivered was the same as in control animals. This critical time course is very different than that seen in trials of low molecular weight chemotherapeutic agents (methotrexate and adriamycin). These preliminary studies suggest that hexosaminidase A can be delivered to the brain by blood--brain barrier modification and may be indicative of the potential for enzyme replacement in patients who hae Tay--Sachs disease.

Animals↗
PubMed1985

Essential role of phospholipase A2 activity in endothelial cell-induced modification of low density lipoprotein.

Previous studies have established that incubation of low density lipoprotein (LDL) with cultured endothelial cells (EC) converts it to a new form (EC-modified LDL) that is now recognized by a specific receptor on macrophages (the acetyl LDL receptor) and is taken up and degraded 3-10 times more rapidly than native LDL (biological modification). The formation of EC-modified LDL depended on generation of free radicals with consequent peroxidation of LDL lipids and was accompanied by extensive hydrolysis of LDL phosphatidylcholine at the 2-position. The present studies show that p-bromophenacyl bromide, a site-specific irreversible inhibitor of phospholipase A2 activity, blocks this hydrolysis and, at the same time, the enhanced macrophage degradation. We show further that during EC modification the apoprotein B of LDL undergoes considerable modification and that this also is prevented by the phospholipase inhibitor. Finally, as reported previously, changes similar to those observed on incubation of LDL with EC can be induced by incubation in the absence of cells but in the presence of a sufficiently high concentration of Cu2+. This also is accompanied by hydrolysis of phosphatidylcholine at the 2-position and breakdown of apoprotein B. These changes are also inhibited by p-bromophenacyl bromide, suggesting the presence of a phospholipase A2 activity associated with LDL as it is isolated. A hypothesis is presented linking lipid peroxidation, phosphatidylcholine hydrolysis, and changes in the LDL apoprotein during EC modification.

Acetophenones↗
PubMed1994

Ribosome binding of DNA analogs of tRNA requires base modifications and supports the "extended anticodon".

The efficiency of translation depends on correct tRNA-ribosome interactions. The ability of chemically synthesized yeast tRNA(Phe) anticodon domains to effectively inhibit the binding of native yeast tRNA(Phe) to poly(U)-programmed Escherichia coli 30S ribosomal subunits was dependent on a Mg(2+)-stabilized stem and an open anticodon loop, both facilitated by base modifications. Analysis of tRNA sequences has revealed that base modifications which negate canonical hydrogen bonding are found in 95% of those tRNA anticodon loop sequences with the potential to form two Watson-Crick base pairs across the loop. Therefore, we postulated that a stable anticodon stem and an open loop are prerequisites for ribosome binding. To test this hypothesis, DNA analogs of the yeast tRNA(Phe) anticodon domain were designed to have modification-induced, Mg(2+)-stabilized stems and open loops. The unmodified DNA analog neither bound to poly(U)-programmed 30S ribosomal subunits nor inhibited the binding of native tRNA(Phe). However, specifically modified DNA analogs did bind to ribosomal subunits and effectively inhibited tRNA(Phe) from binding. Thus, modification-dependent Mg(2+)-stabilized anticodon domain structures with open loops have evolved as the preferred anticodon conformations for ribosome binding.

Anticodon↗
PubMed1995

Reduction of copper, but not iron, by human low density lipoprotein (LDL). Implications for metal ion-dependent oxidative modification of LDL.

Cell-mediated oxidative modification of human low density lipoprotein (LDL), most likely an important early step in atherosclerosis, requires redox active metal ions such as copper or iron. We have previously shown that iron-dependent, in contrast to copper-dependent, oxidative modification of LDL requires superoxide, a physiological reductant. In the present study, we sought to explain these discrepant results. LDL was incubated at 37 degrees C with Cu2+ (10 microM) and bathocuproine (BC, 360 microM), an indicator molecule which specifically complexes Cu+, but not Cu2+. In a time- and concentration-dependent manner, LDL reduced Cu2+ to Cu+. An LDL concentration as low as 10 micrograms of protein/ml (about 20 nM) reduced about 7 microM Cu2+ within 1 h of incubation. Complexation of the Cu+ formed under these conditions with BC significantly inhibited oxidative modification of LDL, as assessed by agarose gel electrophoresis. Preincubation of LDL with N-ethylmaleimide had no effect on the rate and extent of Cu2+ reduction nor LDL oxidation, indicating that free sulfhydryl groups associated with apolipoprotein B are not involved. Addition of either superoxide dismutase or catalase or increasing the alpha-tocopherol content of LDL from 11.8 +/- 3.0 to 24.4 +/- 2.8 nmol/mg of protein also had no significant effect on the kinetics of Cu2+ reduction by LDL. In contrast, incubation of LDL with Fe(3+)-citrate (10 microM) and the indicator bathophenanthroline (BP, 360 microM) resulted in no significant Fe2+ formation, even at LDL concentrations as high as 200 micrograms of protein/ml. However, incubation of LDL with Fe(3+)-citrate and an enzymatic source of superoxide led to rapid formation of Fe2+ and consequent oxidative modification of LDL. Addition of BP inhibited iron-mediated LDL oxidation under these conditions. Our results indicate that reduced metal ions are important mediators of LDL oxidation, and that LDL specifically reduces Cu2+, but not Fe3+. These data, therefore, help explain why copper, in addition to being chemically more reactive, is more potent than iron at mediating LDL oxidation.

Copper↗
PubMed1995

Structure-function relationships in human interleukin-11. Identification of regions involved in activity by chemical modification and site-directed mutagenesis.

Chemical modification approaches combined with site-directed and deletion mutagenesis have been used to identify functionally critical regions/residues of recombinant human IL-11 (rhIL-11). Incubation of rhIL-11 with iodoacetic acid results in specific alkylation of a single methionine residue, Met58, and a 25-fold reduction of in vitro biological activity on mouse plasmacytoma cells. A similar decrease in activity is observed when Met58 is substituted with Ala, Leu, Gln, Glu, or Lys by site-directed mutagenesis. Treatment of rhIL-11 with succinic anhydride leads to modification of the amino-terminal amino group and partial labeling of 2 lysines, Lys41 and Lys98, and to a 3-fold decrease in activity. The activity losses can be attributed to modification of the lysine residues, since the succinyl derivative of the amino terminus is fully active. In addition, carboxyl-terminal deletion mutagenesis studies have demonstrated that removal of the last 4 residues reduces rhIL-11 activity 25-fold, whereas removal of 8 or more amino acids results in an inactive molecule. Based on secondary structure predictions and the location of exon/intron boundaries in the IL-11 genomic structure, we propose a four-helix bundle topology as a structural model for rhIL-11. This model has been tested by limited proteolysis using three side chain-specific endoproteinases. A limited number of protease-sensitive cleavage sites are present in rhIL-11, and all but two are located in the postulated helix interconnecting loops or at helix termini. alpha-Helices, which in the proposed structure form a compact core of the molecule, are inaccessible to digestion under limiting conditions. According to the model, Met58, Lys41 and Lys98 are located on the surface of the molecule, in agreement with their preferential accessibility to chemical modifications. By analogy with human growth hormone, we postulate that Met58 and the carboxyl terminus of rhIL-11 are involved in the primary receptor binding site (site I), whereas Lys41 and Lys98 may be a part of binding site II.

Amino Acid Sequence↗
PubMed1995

Carbonic anhydrase III. Oxidative modification in vivo and loss of phosphatase activity during aging.

Oxidative modification of DNA, lipids, and proteins occurs as a consequence of reaction with free radicals and activated oxygen. Oxidative modification of total cellular proteins has been described under many pathologic and experimental conditions, but no specific proteins have been identified as in vivo targets for oxidative modification. Utilizing an immunochemical method for detection of oxidatively modified proteins, we identified a protein in rat liver that was highly oxidized. It was purified to homogeneity and identified as carbonic anhydrase, isozyme III. Its characteristics match those previously described for a protein that was lost during aging of the rat, senescence marker protein-1. Carbonic anhydrase III was purified from rats aged 2, 10, and 18 months, and the proteins were characterized. All three preparations were highly oxidatively modified as assessed by their carbonyl content. The enzyme has three known catalytic activities, and the specific activities for carbon dioxide hydration and for ester hydrolysis decreased during aging by approximately 30%. However, the third activity, that of a phosphatase, was virtually lost during aging. While the physiologic role of carbonic anhydrase III is unknown, we suggest that it functions in an oxidizing environment, which leads to its own oxidative modification.

Aging↗
PubMed1996

3'-O-(4-Benzoyl)benzoyladenosine 5'-triphosphate inhibits activity of the vacuolar (H+)-ATPase from bovine brain clathrin-coated vesicles by modification of a rapidly exchangeable, noncatalytic nucleotide binding site on the B subunit.

It was previously observed that the B subunit of the tonoplast V-ATPase is modified by the photoactivated nucleotide analog 3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate (BzATP) (Manolson, M. F., Rea, P. A., and Poole, R. J. (1985) J. Biol. Chem. 260, 12273-12279). We have further characterized the nucleotide binding sites on the V-ATPase and the interaction between BzATP and the B subunit. We observe that the V-ATPase isolated from bovine clathrin-coated vesicles possesses approximately 1 mol of endogenous, tightly bound ATP/mol of V-ATPase complex. BzATP is not a substrate for the V-ATPase, but does act as a noncovalent inhibitor in the absence of irradiation, changing the kinetic characteristics of ATP hydrolysis. Irradiation of the V-ATPase in the presence of [3H]BzATP results primarily in modification of the 58-kDa B subunit, with complete inhibition of V-ATPase activity occurring upon modification of one B subunit per V-ATPase complex. Inhibition occurs as the result of modification of a rapidly (t1/2 < 2 min) exchangeable site, and yet this site does not correspond to a catalytic site, as indicated by the effects of cysteine-modifying reagents which react with Cys254 located at the catalytic sites on the A subunit. Thus, the noncatalytic nucleotide binding site modified by BzATP appears to be rapidly exchangeable. The site of [3H]BzATP modification of the B subunit was localized to the region Ile164 to Gln171, which from the x-ray crystal structure of the homologous F-ATPase alpha subunit, is within 10 A of the ribose ring of ATP bound to the noncatalytic nucleotide binding site. Thus, despite the absence of a glycine-rich loop region in the B subunit, these data are consistent with a similar overall folding pattern for the V-ATPase B subunit and the F-ATPase alpha subunit.

Adenosine Triphosphate↗
PubMed1996

Post-translational modifications of Ras and Ral are important for the action of Ral GDP dissociation stimulator.

Ral GDP dissociation stimulator (RalGDS) is a GDP/GTP exchange protein of Ral and a new effector protein of Ras. Therefore, there may be a new signaling pathway from Ras to Ral. In this paper, we examined the roles of the post-translational modifications of Ras and Ral on this new signal transduction pathway. The post-translationally modified form of Ras bound to RalGDS more effectively than the unmodified form. The modification of Ras was required to regulate the distribution of RalGDS between the cytosol and membrane fractions in COS cells. The post-translational modification of Ral enhanced the activities of RalGDS to stimulate the dissociation of GDP from and the binding of GTP to Ral. Furthermore, the modified form of Ral bound to Ral-binding protein 1 (RalBP1), a putative effector protein of Ral, more effectively than the unmodified form. Taken together with the observations that Ras and Ral are localized to the membranes, these results suggest that the post-translational modifications of Ras and Ral play a role for transmitting the signal effectively on the membranes in the signal transduction pathway of Ras/RalGDS/Ral/RalBP1.

Amino Acid Sequence↗
PubMed1996

Chemical modification and site-directed mutagenesis of cysteine residues in human placental S-adenosylhomocysteine hydrolase.

Human placental S-adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1. 1) was inactivated by 5',5-dithiobis(2-nitrobenzoic acid) following pseudo-first-order kinetics. Modification of three of the 10 cysteine residues per enzyme subunit resulted in complete inactivation of the enzyme. The three modified cysteine residues were identified as Cys113, Cys195, and Cys421, respectively, by protein sequencing after modification with [1-14C]iodoacetamide. Of the three modifiable cysteines, Cys113 and Cys195 could be protected from modification in the presence of the substrate adenosine (Ado), which also protected the enzyme from inactivation. On the other hand, Cys421 was not protected by Ado, and modification of Cys421 alone did not affect the enzyme activity. To verify whether some of these cysteine residues are important for the enzyme catalysis, these three cysteine residues were replaced by either serine or aspartic acid using site-directed mutagenesis. Mutants of both Cys113 (C113S and C113D) and Cys421 (C421S and C421D) had enzyme activities similar to that of the wild-type enzyme, and only slight changes were observed in the steady-state kinetics measured in both the synthetic and hydrolytic directions. However, mutants of Cys195 (C195D and C195S) displayed drastically reduced enzyme activities, and kcat values were only 7 and 12% of that of the wild-type enzyme, respectively, resulting in a calculated loss in binding energy (DeltaDeltaG) of approximate 1 Kcal/mol. The Cys195 mutants were capable of catalyzing both the 3'-oxidative and 5'-hydrolytic reactions, as evidenced by the reduction of E.NAD+ to NADH and formation of the 5'-hydrolytic product when incubated with (E)-5', 6'-didehydro-6'-deoxy-6'-chlorohomoadenosine at rates comparable with those catalyzed by the wild-type enzyme. However, mutations of the Cys195 severely altered the 3'-reduction potential as evidenced by the drastic reduction in the rate of [2,8-3H]Ado release from the E-NADH.[2,8-3H]3'-keto-Ado complex. Circular dichroism studies of the Cys195 mutants confirmed that the observed effects are not due to changes in secondary structure. These results suggested that the Cys195 is involved in the catalytic center and may play an important role in maintaining the 3'-reduction potential for effective release of the reaction products and regeneration of the active form (NAD+ form) of the enzyme; the Cys113 is located in or near the substrate binding site, but plays no role beneficial to the catalysis; and the Cys421 is a nonessential residue, which also explains why Cys421 does not occur in any other known AdoHcy hydrolases.

Adenosylhomocysteinase↗
PubMed1997

Oxidation of low density lipoprotein particles decreases their ability to bind to human aortic proteoglycans. Dependence on oxidative modification of the lysine residues.

Oxidation of low density lipoprotein (LDL) leads to its rapid uptake by macrophages in vitro, but no detailed studies have addressed the effect of oxidation on the binding of LDL to proteoglycans. We therefore treated LDL with various substances: copper sulfate, 2,2'-azobis(2-amidinopropane)hydrochloride (AAPH), soybean lipoxygenase, and mouse peritoneal macrophages, and determined the extent to which the oxidatively modified LDL bound to human aortic proteoglycans in an affinity column. Oxidation of LDL with copper, AAPH, or macrophages, all of which increased its electrophoretic mobility, was associated with reduced binding to proteoglycans, until strongly oxidized LDL was totally unable to bind to them. After treatment of LDL with soybean lipoxygenase, the change in electrophoretic mobility was small, and the amount of binding to proteoglycans was only slightly decreased. The increased electrophoretic mobility of oxidized LDL reflects modification of the lysine residues of apolipoprotein B-100 (apoB-100). To mimic the oxidative modification of lysines, we treated LDL with malondialdehyde. This treatment also totally prevented the binding of LDL to proteoglycans. In contrast, if the lysine residues of apoB-100 were methylated to shield them against oxidative modification, subsequent treatment of LDL with copper sulfate failed to reduce the degree of LDL binding to proteoglycans. Finally, the active lysine residues in the oxidized LDL particles, which are thought to be involved in this binding, were quantified with NMR spectroscopy. In oxidized LDL, the number of these residues was found to be decreased. The present results show that, after modification of the lysine residues of apoB-100 during oxidation, the binding of LDL to proteoglycans is decreased, and suggest that oxidation of LDL tends to lead to intracellular rather than extracellular accumulation of LDL during atherogenesis.

Animals↗
PubMed1997

Identification of modification sites in large biomolecules by stable isotope labeling and tandem high resolution mass spectrometry. The active site nucleophile of thiaminase I.

A widely used procedure for site localization of covalent protein modifications involves proteolysis, partial chromatographic separation of the resulting complex mixture, and tandem mass spectrometry (MS/MS) to identify peptides whose molecular weight (Mr) has been increased appropriately by the modification. As found previously for MS of small molecules, this study shows that protein fragment identification can be greatly simplified by labeling the modification with stable isotopes. Further, the high resolution capabilities of Fourier transform MS make possible the direct identification of CH3/CD3-labeled peptides without chromatographic separation. Although separate Asp-N, Lys-C, and alpha-chymotrypsin digests of thiaminase I (42 kDa) yielded as many as 70 peptides, FTMS identification of the labeled peptide localized the modification site of a mechanism-based inhibitor to Arg101-Lys121, Asp90-Gly122, and Gly107-Tyr119, respectively. The measured mass difference values of the two labels agreed with that expected for CH3/CD3, 3.019 Da, with a standard deviation of 0.005 Da, providing persuasive identity verification. MS/MS fragmentation narrowed the site to Pro109-Phe118 and also caused loss of the derivative with a sulfur atom, uniquely identifying Cys113 as the thiaminase I active-site nucleophile among the 379 amino acids.

Alkyl and Aryl Transferases↗
PubMed1998

Immunological evidence for methylglyoxal-derived modifications in vivo. Determination of antigenic epitopes.

The Maillard reaction, a non-enzymatic reaction of ketones and aldehydes with amino groups of proteins, contributes to the aging of proteins and to complications associated with diabetes. Methylglyoxal (MG) is a 2-oxoaldehyde derived from glycolytic intermediates and produced during the Maillard reaction. We reported previously the formation of a lysine-lysine protein cross-linking structure (imidazolysine) and a fluorescent arginine modification (argpyrimidine) from the Maillard reaction of MG. Here we show that rabbit antibodies to MG-modified ribonuclease A identify proteins modified by the Maillard reaction of glucose, fructose, ribose, glyceraldehyde, glyoxal, ascorbate, and ascorbate oxidation products (dehydroascorbate, 2,3-diketogulonate, L-xylosone, and L-threose) in addition to those modified by MG. The antibody recognized imidazolysine and argpyrimidine and a glyoxal-derived lysine-lysine cross-link. It did not react with Nepsilon-carboxymethyllysine. Incubations with amino acids revealed strongest reactivity with Nalpha-t-butoxycarbonylarginine and MG, and we identified argpyrimidine as one of the epitopes from this incubation mixture. Serum proteins from human diabetics reacted more strongly with the antibody than those from normal individuals, and the levels correlated with glycemic control. Collagen from human corneas contained MG-derived modifications, with those from older subjects containing higher levels of modified proteins than those from younger ones. An immunoaffinity-purified antibody showed higher reactivity with old corneas than with younger ones and localized the antigens primarily within the stromal region of the cornea. These results confirm reported MG-derived modifications in tissue proteins and show that dicarbonyl-mediated protein modification occurs during Maillard reactions in vivo.

Adult↗
PubMed2001

Oncogenic Ki-ras confers a more aggressive colon cancer phenotype through modification of transforming growth factor-beta receptor III.

Transforming growth factor-beta1 (TGF-beta1) can act as a tumor suppressor or a tumor promoter depending on the characteristics of the malignant cell. Each of three Ki-ras(G12V) transfectants of HD6-4 colon cancer cells had been shown to be more aggressive in vivo than controls in earlier studies (Yan, Z., Chen, M., Perucho, M., and Friedman, E. (1997) J. Biol. Chem. 272, 30928-30936). We now show that stable expression of oncogenic Ki-ras(G12V) converts the HD6-4 colon cancer cell line from insensitive to TGF-beta1 to growth-promoted by TGF-beta1. Each of three Ki-ras(G12V) transfectants responded to TGF-beta1 by an increase in proliferation and by decreasing the abundance of the Cdk inhibitor p21 and the tumor suppressor PTEN, whereas each of three wild-type Ki-ras transfectants remained unresponsive to TGF-beta1. The wild-type Ki-ras transfectants lack functional TGF-beta receptors, whereas all three Ki-ras(G12V) transfectants expressed functional TGF-beta receptors that bound (125)I-TGF-beta1. The previous studies showed that in cells with wild-type Ki-ras, TGF-beta receptors were not mutated, and receptor proteins were transported to the cell surface, but post-translational modification of TGF-beta receptor III (TbetaRIII) was incomplete. We now show that the betaglycan form of TbetaRIII is highly modified following translation when transiently expressed in Ki-ras(G12V) cells, whereas no such post-translational modification of TbetaRIII occurs in control cells. Antisense oligonucleotides directed to Ki-Ras decreased both TbetaRIII post-translational modification in Ki-ras(G12V) cells and TGF-beta1 down-regulation of p21, demonstrating the direct effect of mutant Ras. Therefore, one mechanism by which mutant Ki-Ras confers a more aggressive tumor phenotype is by enhancing TbetaRIII post-translational modification.

Activin Receptors, Type I↗
PubMed2001

Ligand-selective modulation of the permeability transition pore by arginine modification. Opposing effects of p-hydroxyphenylglyoxal and phenylglyoxal.

Chemical modification of mitochondria with the arginine-specific reagents phenylglyoxal (PGO) and 2,3-butanedione (BAD) decreases the Ca(2+) sensitivity of the permeability transition pore (PTP) and stabilizes it in the closed conformation (Eriksson, O., Fontaine, E., and Bernardi, P. (1998) J. Biol. Chem. 273, 12669-12674). Unexpectedly, modification of mitochondria with the arginine-specific reagent p-hydroxyphenylglyoxal (OH-PGO) resulted instead in PTP opening. Sequential modification with OH-PGO and PGO (or BAD) revealed that the effects on the PTP depended on the order of the additions. PTP opening was observed when OH-PGO preceded, and PTP closing was observed when OH-PGO followed, the addition of PGO (or BAD). The differential effects of OH-PGO and PGO on the PTP open probability (i) were not modified by the conformation-specific ligands of the adenine nucleotide translocase bongkrekate and atractylate; and (ii) were also observed in de-energized mitochondria, indicating that the effect is exerted directly on the PTP. OH-PGO dramatically sensitized PTP opening, which was triggered by depolarization even in the presence of EGTA. These data show that arginine modification modulates the PTP conformation in a ligand-selective fashion and suggest that the effects of OH-PGO, PGO, and BAD are mediated by the same arginine residues. We analyzed the structure of the arginine adducts by matrix-assisted laser desorption ionization and time-of-flight mass spectrometry using a test peptide and N-acetylarginine. The results indicate that both OH-PGO and PGO react with arginine at a stoichiometry of 2:1 and form stable adducts that may be feasible to identify the PTP at the molecular level.

Animals↗
PubMed2001

Active site residues of cephalosporin acylase are critical not only for enzymatic catalysis but also for post-translational modification.

Cephalosporin acylase (CA) is a recently identified N-terminal hydrolase. It is also a commercially important enzyme in producing 7-aminocephalosporanic acid (7-ACA), a backbone chemical in synthesizing semi-synthetic cephalosporin antibiotics. CA is translated as an inactive single chain precursor, being post-translationally modified into an active enzyme. The post-translational modification takes place in two steps. The first intramolecular autocatalytic proteolysis takes place at one end of the spacer peptide by a nucleophilic Ser or Thr, which in turn becomes a new N-terminal Ser or Thr. The second intermolecular modification cleaves off the other end of the spacer peptide by another CA. Two binary structures in complex with glutaryl-7-ACA (the most favored substrate of CAs) and glutarate (side chain of glutaryl-7-ACA) were determined, and they revealed the detailed interactions of glutaryl-7-ACA with the active site residues (Y. Kim and W. G. J. Hol (2001) Chem. Biol., in press). In this report: 1) we have mutated key active site residues into nonfunctional amino acids, and their roles in catalysis were further analyzed; 2) we performed mutagenesis studies indicating that secondary intermolecular modification is carried out in the same active site where deacylation reaction of CA occurs; and 3) the cleavage site of secondary intermolecular modification by another CA was identified in the spacer peptide using mutational analysis. Finally, a schematic model for intermolecular cleavage of CA is proposed.

Acylation↗