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At least 613 records · Page 34Linked to original sources
PubMed2003

Identification and characterization of mouse MTO1 gene related to mitochondrial tRNA modification.

The nucleotide modification in tRNA plays a pivotal role in the fidelity of translational process. The defects in nucleotide modification have often been observed in the mutated mitochondrial tRNAs associated with human diseases. Recently, MTO1-like protein in bacteria and yeast has been implicated to be a component of tRNA modification pathway. Here we report the identification and characterization of mouse MTO1 homolog. The mouse MTO1 gene containing 12 exons encodes a 669-residue protein with a strong homology to the MTO1-like proteins of bacteria and yeast, related to tRNA modification. Functional conservation of this protein is supported by the observation that the isolated mouse MTO1 cDNA can complement the respiratory-deficient phenotype of yeast mto1 cells carrying P(R)(454) mutation. MTO1 is ubiquitously expressed in various tissues, but with markedly elevated expression in tissues of high metabolic rates. Furthermore, we showed that mouse Mto1 localizes in mitochondrion. These observations suggest that the mouse MTO1 is a structural and functional homolog of yeast MTO1, thereby playing a role in the mitochondrial tRNA modification and protein synthesis.

Animals↗
PubMed2003

Group V and X secretory phospholipase A(2)s-induced modification of high-density lipoprotein linked to the reduction of its antiatherogenic functions.

The quantitative or qualitative decline of high-density lipoprotein (HDL) is linked to the pathogenesis of atherosclerosis because of its antiatherogenic functions, including the mediation of reverse cholesterol transport from the peripheral cells to the liver. We have recently shown that group X secretory phospholipase A(2) (sPLA(2)-X) is involved in the pathogenesis of atherosclerosis via potent lipolysis of low-density lipoprotein (LDL) leading to macrophage foam cell formation. We demonstrate here that sPLA(2)-X as well as group V secretory PLA(2) (sPLA(2)-V), another group of sPLA(2) that can potently hydrolyze phosphatidylcholine (PC), also possess potent hydrolytic potency for PC in HDL linked to the production of a large amount of unsaturated fatty acids and lysophosphatidylcholine (lysoPC). In contrast, the classical types of group IB and IIA secretory PLA(2)s evoked little, if any, lypolytic modification of HDL. Treatment with sPLA(2)-X or -V also caused an increase in the negative charge of HDL with no oxidation and little modification of apolipoprotein AI (apoAI). Modification with sPLA(2)-X or -V resulted in significant decrease in the capacity of HDL to cause cellular cholesterol efflux from lipid-loaded macrophages. Immunohistochemical analysis revealed significant expression of sPLA(2)-X in foam cell lesions in the arterial intima of Watanabe heritable hyperlipidemic (WHHL) rabbit. These findings suggest that lipolytic modification of HDL by sPLA(2)-X or -V causes drastic change of HDL in terms of the production of a large amount of unsaturated fatty acids and lysoPC linked to the reduction of its antiatherogenic functions. These sPLA(2)-mediated modifications of plasma lipoproteins might be relevant to the pathogenesis of atherosclerosis.

Animals↗
PubMed2000

Interrelated modification of excitatory and inhibitory synapses in three-layer olivary-cerebellar neural network.

The model of three-layer olivary-cerebellar neural network with modifiable excitatory and inhibitory connections between diverse elements is suggested. The same Hebbian modification rules are proposed for Purkinje cells, granule (input) cells, and deep cerebellar nuclei (output) cells. The inverse calcium-dependent modification rules for these cells and hippocampal/neocortical neurones or Golgi cells are conceivably the result of the involvement of cGMP and cAMP in postsynaptic processes. The sign of simultaneous modification of excitatory and inhibitory inputs to a cell is opposite and determined by the variations in pre- and/or postsynaptic cell activity. Modification of excitatory transmission between parallel fibers and Purkinje cells, mossy fibers and granule cells, and mossy fibers and deep cerebellar nuclei cells essentially depends on inhibition effected by stellate/basket cells, Golgi cells and Purkinje cells, respectively. The character of interrelated modifications of diverse synapses in all three layers of the network is influenced by olivary cell activity. In the absence (presence) of a signal from inferior olive, the long-term potentiation (depression) in the efficacy of a synapse between input mossy fiber and output cell can be induced. The results of the suggested model are in accordance with known experimental data.

Animals↗
PubMed2002

Characterization of the O-GlcNAc protein modification in Xenopus laevis oocyte during oogenesis and progesterone-stimulated maturation.

Little information exists about single N-acetylglucosamine modifications on proteins in growth and developmental model systems. To explore these phenomena, Xenopus laevis oocytes from stages I-VI of oogenesis were isolated and proteins analyzed on SDS-PAGE. The proteins were probed with antibodies specific for O-GlcNAc. Levels of the O-GlcNAc protein modification were highest in stages I and II, while decreasing in stages III-VI. The reduction in amount of O-GlcNAc-modified proteins was correlated to increases in apparent O-GlcNAcase (streptozotocin-inhibitable neutral hexosaminidase), activity involved in removing protein monoglycosylations. The O-GlcNAc modification was also characterized during progesterone-stimulated oocyte maturation. Although O-GlcNAcase activity appeared relatively constant between quiescent and matured stage VI oocytes, a small decrease in the levels of both total and specific O-GlcNAc-modified proteins was observed. Investigating the function of O-GlcNAc during maturation, oocytes were incubated with compounds known to modulate the levels of the O-GlcNAc protein modification and then stimulated to mature. Oocytes treated with compounds known to increase O-glycosylation consistently matured slower than non-treated controls, while oocytes treated with compounds that decrease O-glycosylation matured slightly faster than controls. The O-GlcNAc modification may play important roles in both the developmental and cell division processes of X. laevis oocytes.

Acetylglucosamine↗
PubMed1997

Influence of histone acetylation on the modification of cytoplasmic and nuclear proteins by ADP-ribosylation in response to free radicals.

Inhibition of histone deacetylase by addition of 5 mM n-sodium butyrate to the growth medium increases the utilization of [32P]NAD+ and ADP-ribosylation (ADPR) of total cellular proteins of V79, HeLa, mouse B16, mouse Fib/T and human T1 kidney cells by a factor of 1.2-2.3. When the ADP-ribosylase is challenged by exposing cells to damage by .OH radicals (25 microM CuSO4 2.8 mM H2O2) ADPR increases by factors of 5.7-6.0 and 3.2-4.0 in normal and butyrated cells, respectively. Operation of the free radical generator is supported by the response to EDTA and radical scavengers. Densitometric analysis of autoradiographs from SDS-gels show that butyrate exposure increases basal ADPR-modification of histones from T1 cells by factors of 1.1-1.9. Addition of .OH radicals increases the ADPR modifications of histones 4.4-8.7-fold in normal cells and 3.2-6.7-fold in butyrate exposed cells. Butyrate exposure elevates base level ADPR-modification and reduces subsequent ADPR-modification initiated by DNA damage. The results are consistent with the view that ADPR-modification and histone acetylation have overlapping functions and probably induce similar structural changes in chromatin.

Acetylation↗
PubMed1997

Protein thiol modifications of human red blood cells treated with t-butyl hydroperoxide.

Oxidative stress causes modification of cellular macromolecules and leads to cell damage. The objective of this study was to identify protein modifications that relate to thiol groups in human red blood cells under oxidative stress. With t-butyl hydroperoxide (t-BH) treatment, results of isoelectric focusing (IEF) analysis showed that two dithiothreitol-reversible modifications are observed, one toward the cathode and the other to the anode. Protein change toward the cathode was demonstrated to be hemoglobin oxidation, which gains a net positive charge, based on the same focus on IEF gels as hemoglobin and methemoglobin and molecular weight analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Otherwise, the change toward the anode was the result of mixed disulfide formation between GSH and protein thiols. Based on the results of molecular weight analysis and its reversion from methemoglobin, protein formed mixed disulfides with GSH were also regarded as hemoglobin. As red blood samples were treated with diamide or GSSG, in addition to the mixed disulfides observed in t-BH-treated cells, additional hemoglobin-GSH mixed disulfide appeared. But the disappearance of this diamide-induced additional mixed disulfide by treating cells with t-BH after diamide treatment suggests that the increase of negative charges from GSH are offset by ferrohemoglobin oxidation to ferrihemoglobin. Additionally, other dithiothreitol-reversible modifications of one cell membrane protein, spectrin, were also observed from the formation of high molecular weight molecules as detected by SDS-PAGE. Results indicate that protein thiols in human red blood cells are susceptible to modification under oxidative stress. IEF analysis provides a useful tool to measure methemoglobin and hemoglobin GSH mixed disulfide formation.

Adult↗
PubMed1998

Comparisons of quality of life and cardiac performance after complete atrioventricular junction ablation and atrioventricular junction modification in patients with medically refractory atrial fibrillation.

OBJECTIVES: This study compared the long-term effects of complete atrioventricular junction (AVJ) ablation with those of AVJ modification in patients with medically refractory atrial fibrillation (AF). BACKGROUND: Comparisons between the long-term effects of AVJ ablation with those of AVJ modification in patients with medically refractory AF have not been systematically studied. METHODS: Sixty patients with medically refractory AF were randomly assigned to receive complete AVJ ablation with permanent pacing or AVJ modification. Subjective perception of quality of life (QOL) was assessed by a semiquantitative questionnaire before and 1 and 6 months after ablation. Cardiac performance was evaluated by echocardiography and radionuclide angiography within 24 h (baseline) and at 1 and 6 months after ablation. RESULTS: Both methods were associated with significant improvement in general QOL and a significant reduction in the frequency of major symptoms and symptoms during attacks. The frequency of hospital admission and emergency room visits and antiarrhythmic drug trials significantly decreased after ablation in both groups. However, patients after complete AVJ ablation had a significantly greater improvement in general QOL and a significantly reduced frequency of major symptoms and symptoms during attacks (including palpitation, dizziness, chest oppression, blurred vision and syncope). Left ventricular (LV) systolic function and the ability to perform activities of daily life significantly improved after ablation in patients with depressed LV function in both groups. All improvements after ablation or modification were maintained over the 6-month follow-up period. CONCLUSIONS: AVJ ablation with permanent pacing, as compared with AVJ modification, had a significantly greater ability to decrease the frequency of attacks and the extent of symptoms of AF, and the patients who received this procedure were more satisfied with their general well-being.

Activities of Daily Living↗
PubMed1997

Modification of proteins in endothelial cell death during oxidative stress.

Exposure of bovine aortic endothelial cells in vitro to oxidative stress causes a cascade of changes in cell function, culminating in cell death if the stress is sufficiently severe. Oxidative modification of proteins, as measured by the reaction of 2,4-dinitrophenylhydrazine with carbonyl groups of oxidized proteins, increased three- to fourfold in endothelial cells exposed to hydrogen peroxide or to a xanthine/xanthine oxidase system. The increase in oxidative modification of protein occurred rapidly, preceding loss of cellular ATP and eventual cell death. Oxidative modification of protein was paralleled by loss of activity of the key metabolic enzymes, glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. The finding that oxidative modification of protein is an early event following oxidative stress suggests that oxidative modification of protein is not only a marker for oxidative damage but also a causal factor in oxidative injury.

Adenosine Triphosphate↗
PubMed1999

A systematic approach to evaluate the modification of lens proteins by glycation-induced crosslinking.

To systematically evaluate the modification of lens proteins by aldose and dicarbonyl sugars during the glycation process, the sugar-dependent incorporation of Lys and Arg, SDS-PAGE profile, amino acid analysis, and fluorophore formation (excitation 370 nm/emission 440 nm) were determined. Reaction mixtures with glycolaldehyde, glyceraldehyde, threose and 3-deoxythreosone showed the greatest extent of Lys crosslinking and fluorescence formation. An increase in fluorescence intensity, but a decrease in Lys and Arg crosslinking, was found with glyoxal, methylglyoxal, hydroxypyruvaldehyde and threosone. In addition glyoxal, methylglyoxal and hydroxypyruvaldehyde caused the specific loss of Arg residues in lens proteins. Reaction mixtures with xylose, xylosone, glucose, glucosone and 3-deoxyglucosone exhibited the least protein modifications; however, incubation with 3-deoxyxylosone resulted in extensive loss of Lys and Arg residues, a higher extent of Lys or Arg crosslinking and significant fluorophore formation. Each sugar exhibited unique characteristics in the modification of lens proteins by glycation. To validly compare the protein modifications occurring during glycation reactions, a systematic approach was employed to evaluate the potential role of aldose and dicarbonyl sugars in protein modification.

Arginine↗
PubMed2002

Non-enzymatic covalent modifications of proteins: mechanisms, physiological consequences and clinical applications.

Given the complexity of the biosynthetic machinery and the delicate chemical composition of proteins, it is remarkable that cells manage to produce and maintain normally functioning proteins under most conditions. However, it is now well known that proteins are susceptible to various non-enzymatic covalent modifications (NECM) under physiological conditions. Such modifications can be of no or little importance to the protein or they can be absolutely detrimental. Often NECM are difficult to study due to the complex and technically demanding methods required to identify many of these modifications. Thus, the role of NECM has not yet been adequately resolved but recent research has allowed a better understanding of such modifications. The present review outlines the various forms of NECM that involve covalent modifications of proteins, and discusses their relevance, biological impact and potential applications in the study of protein turnover and diagnosis of disease.

Animals↗
PubMed1999

Chemical modification of enzymes for enhanced functionality.

The explosion in commercial and synthetic applications of enzymes has stimulated much of the interest in enhancing enzyme functionality and stability. Covalent chemical modification, the original method available for altering protein properties, has now re-emerged as a powerful complementary approach to site-directed mutagenesis and directed evolution for tailoring proteins and enzymes. Glutaraldehyde crosslinking of enzyme crystals and polyethylene glycol (PEG) modification of enzyme surface amino groups are practical methods to enhance biocatalyst stability. Whereas crosslinking of enzyme crystals generates easily recoverable insoluble biocatalysts, PEGylation increases solubility in organic solvents. Chemical modification has been exploited for the incorporation of cofactors onto protein templates and for atom replacement in order to generate new functionality, such as the conversion of a hydrolase into a peroxidase. Despite the breadth of applicability of chemically modified enzymes, a difficulty that has previously impeded their implementation is the lack of chemo- or regio-specificity of chemical modifications, which can yield heterogeneous and irreproducible product mixtures. This challenge has recently been addressed by the introduction of a unique position for modification by a site-directed mutation that can subsequently be chemically modified to introduce an unnatural amino acid sidechain in a highly chemo- and regio-specific manner.

Biotechnology↗
PubMed1998

Birth of the snoRNPs: the evolution of the modification-guide snoRNAs.

Bacteria and eukaryotes adopt very different strategies to modify their rRNAs. Most sites of eukaryotic rRNA modification are selected by guide small nucleolar RNAs (snoRNAs), while bacteria rely on numerous site-specific modification enzymes. This raises a 'chicken and egg' dilemma: how could a system of modification that requires a large number of snoRNA cofactors have developed? Did it arise in a de novo fashion, or evolve from a pre-existing protein-based system? The rRNA sequences are well conserved in evolution, but the pattern of modification is only moderately conserved, and many more sites are modified in eukaryotes than in bacteria; why is this so? We propose a model for the origins of the modification-guide snoRNAs that attempts to answer these questions.

Cell Nucleolus↗
PubMed2002

The SUMO-1 isopeptidase Smt4 is linked to centromeric cohesion through SUMO-1 modification of DNA topoisomerase II.

In S. cerevisiae, posttranslational modification by the ubiquitin-like Smt3/SUMO-1 protein is essential for survival, but functions and cellular targets for this modification are largely unknown. We find that one function associated with the Smt3/SUMO-1 isopeptidase Smt4 is to control chromosome cohesion at centromeric regions and that a key Smt3/SUMO-1 substrate underlying this function is Top2, DNA Topoisomerase II. Top2 modification by Smt3/SUMO-1 is misregulated in smt4 strains, and top2 mutants resistant to Smt3/SUMO-1 modification suppress the smt4 cohesion defect. top2 mutants display aberrant chromatid stretching at the centromere in response to mitotic spindle tension and altered chromatid reassociation following microtubule depolymerization. These results suggest Top2 modification by Smt3/SUMO-1 regulates a component of chromatin structure or topology required for centromeric cohesion.

Cell Cycle Proteins↗
PubMed1999

Lipid modifications of proteins - slipping in and out of membranes.

Protein lipid modification, once thought to act as a stable membrane anchor for soluble proteins, is now attracting more widespread attention for its emerging role in diverse signaling pathways and regulatory mechanisms. Most multicellular organisms have recruited specific types of lipids and a suite of unique enzymes to catalyze the modification of a select number of proteins, many of which are evolutionarily conserved in plants, animals and fungi. Each of the three known types of lipid modification - palmitoylation, myristylation and prenylation - allows cells to target proteins to the plasma membrane, as well as to other subcellular compartments. Among the lipid modifications, protein prenylation might also function as a relay between cytoplasmic isoprene biosynthesis and regulatory pathways that control cell cycle and growth. Molecular and genetic studies of an Arabidopsis mutant that lacks farnesyl transferase suggest that the enzyme has a role in abscisic acid signaling during seed germination and in the stomata. It is becoming clear that lipid modifications are not just fat for the protein, but part of a highly conserved intricate network that plays a role in coordinating complex cellular functions.

Journal Article↗
PubMed2004

Intra-specific host sharing in the manipulative parasite Acanthocephalus dirus: does conflict occur over host modification?

The acanthocephalan parasite Acanthocephalus dirus induces a colour change in the intermediate host, the aquatic isopod Caecidotea intermedius, which increases transmission to definitive hosts (creek chub, sunfish). We examined the potential for conflict to occur between infective (cystacanth) and non-infective (acanthor, acanthella) stages of A. dirus over the level of colour modification that should be induced when these stages share a host. Using a field survey, we showed that host sharing by infective and non-infective stages was relatively common and that infective and non-infective stages differed in their effects on colour modification. Non-infective stages induced a colour change over 40% of the body, whereas infective stages induced a colour change over 80%. Thus, conflict could occur between stages over the level of modification that should be induced. We then showed that mixed-stage infections induced a colour change in the host that was consistent with the level of modification induced by the infective stage. We discuss the potential significance of these results to patterns of host modification and their effects on stage-related survival in nature.

Acanthocephala↗
PubMed2000

Probing the structure of monomers and dimers of the bacterial virus phi29 hexamer RNA complex by chemical modification.

All dsDNA viruses multiply their genome and assemble a procapsid, a protein shell devoid of DNA. The genome is subsequently inserted into the procapsid. The bacterial virus phi29 DNA translocating motor contains a hexameric RNA complex composed of six pRNAs. Recently, we found that pRNA dimers are building blocks of pRNA hexamers. Here, we report the structural probing of pRNA monomers and dimers by chemical modification under native conditions and in the presence or absence of Mg2+. The chemical-modification pattern of the monomer is compared to that of the dimer. The data strongly support the previous secondary-structure prediction of the pRNA concerning the single-stranded areas, including three loops and seven bulges. However, discrepancies between the modification patterns of two predicted helical regions suggest the presence of more complicated, higher-order structure in these areas. It was found that dimers were formed via hand-in-hand and head-to-head contact, as the interacting sequence of the right and left loops and all bases in the head loop were protected from chemical modification. Cryoatomic force microscopy revealed that the monomer displayed a check-mark shape and the dimer exhibited an elongated shape. The dimer was twice as long as the monomer. Direct observation of the shape and measurement of size and thickness of the images strongly support the conclusion from chemical modification concerning the head-to-head contact in dimer formation. Our results also suggest that the role for Mg2+ in pRNA folding is to generate a proper configuration for the right and head loops, which play key roles in this symmetrical head-to-head organization. This explains why Mg2+ plays a critical role in pRNA dimer formation, procapsid binding, and phi29 DNA packaging.

Bacillus Phages↗
PubMed2003

Complete characterization of posttranslational modification sites in the bovine milk protein PP3 by tandem mass spectrometry with electron capture dissociation as the last stage.

A comprehensive approach to protein identification and determination of sites of posttranslational modifications (PTMs) in heavily modified proteins was tested. In this approach, termed "reconstructed molecular mass analysis" (REMMA), the molecular mass distribution of the intact protein is measured first, which reveals the extent and heterogeneity of modifications. Then the protein is digested with one or several enzymes, with peptides separated by reversed-phase HPLC, and analyzed by Fourier transform mass spectrometry (FTMS). Vibrational excitation (collisional or infrared) or electron capture dissociation (ECD) of peptide ions provides protein identification. When a measured peptide molecular mass indicates the possibility of a PTM, vibrational excitation is applied to determine via characteristic losses the type and eventually the structure of the modification, while ECD determines the PTM site. Chromatographic peak analysis continues until full sequence coverage is obtained, after which the molecular mass is reconstructed and compared with the measured value. An agreement indicates that the PTM characterization was complete. This procedure applied to the bovine milk PP3 protein containing 25% modifications by weight yielded all known modifications (five phosphorylations, two O- and one N-glycosylation) as well as the previously unreported NeuNAc-Hex-[NeuNAc]HexNAc group O-linked to Ser60. With the FTMS performance improved, REMMA can serve as the basis for high-throughput, high-sensitivity PTM characterization of biological important proteins, which should speed up the proteomics research.

Amino Acid Sequence↗
PubMed2006

Identification of rotenone-induced modifications in alpha-synuclein using affinity pull-down and tandem mass spectrometry.

Parkinson's disease is a movement disorder that results from a loss of dopaminergic neurons in the substantia nigra. The disease is characterized by mitochondrial dysfunction, oxidative stress, and the presence of "Lewy body" inclusions enriched with aggregated forms of alpha-synuclein, a presynaptic protein. Although alpha-synuclein is modified at various sites in Lewy bodies, it is unclear how sequence-specific posttranslational modifications modulate the aggregation of the protein in oxidatively stressed neurons. To begin to address this problem, we developed an affinity pull-down/mass spectrometry method to characterize the primary structure of histidine-tagged alpha-synuclein isolated from catecholaminergic neurons. Using this method, we mapped posttranslational modifications of alpha-synuclein from untreated neurons and neurons exposed to rotenone, an inhibitor of mitochondrial complex I. Various posttranslational modifications suggestive of oxidative damage or repair were identified in a region comprising a 20-residue stretch in the C-terminal part of the protein. The results indicate that alpha-synuclein is subject to discrete posttranslational modifications in neurons with impaired mitochondrial function. Our affinity pull-down/mass spectrometry method is a useful tool to examine how specific modifications of alpha-synuclein contribute to neurologic disorders such as Parkinson's disease.

Amino Acid Sequence↗