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PubMed2004

A novel partial modification at C2501 in Escherichia coli 23S ribosomal RNA.

Escherichia coli is the best-characterized organism with respect to posttranscriptional modifications of its ribosomal RNA (rRNA). It is presently believed that all the modified nucleotides have been identified, primarily on the basis of two detection methods; modification-induced inhibition of the enzyme reverse transcriptase or analysis by combined HPLC and electrospray ionization mass spectrometry. Comparison of data from these different approaches reveals a disagreement regarding modification of C2501 in E. coli 23S rRNA. A. Bakin and J. Ofengand previously reported the detection of a modification at this site based on a reverse transcriptase assay. J.A. McCloskey and coworkers could not confirm the existence of such a modification using an electrospray ionization mass spectrometry approach. C2501 is therefore generally considered unmodified. We have used a strategy involving isolation of a specific rRNA fragment from E. coli 23S rRNA followed by Matrix Assisted Laser Desorption/Ionization mass spectrometry and tandem mass spectrometry to investigate this controversy. Our data reveal a novel 16-Da partial modification at C2501. We believe that the data reported here clarify the above discrepancy, because a minor partial modification detected in a reverse transcriptase assay would not necessarily be detected by the original mass spectrometry approach. The level of modification was furthermore monitored in different growth situations, and we found a significant positive regulation in stationary phase cells. C2501 is universally conserved and implicated in structure folds very close to the catalytic center of the ribosome. Moreover, several antibiotics bind to nucleotides in this region, which altogether make a modification at this site interesting.

Base Sequence↗
PubMed2005

A genome-wide analysis of C/D and H/ACA-like small nucleolar RNAs in Trypanosoma brucei reveals a trypanosome-specific pattern of rRNA modification.

Small nucleolar RNAs (snoRNAs) constitute newly discovered noncoding small RNAs, most of which function in guiding modifications such as 2'-O-ribose methylation and pseudouridylation on rRNAs and snRNAs. To investigate the genome organization of Trypanosoma brucei snoRNAs and the pattern of rRNA modifications, we used a whole-genome approach to identify the repertoire of these guide RNAs. Twenty-one clusters encoding for 57 C/D snoRNAs and 34 H/ACA-like RNAs, which have the potential to direct 84 methylations and 32 pseudouridines, respectively, were identified. The number of 2'-O-methyls (Nms) identified on rRNA represent 80% of the expected modifications. The modifications guided by these RNAs suggest that trypanosomes contain many modifications and guide RNAs relative to their genome size. Interestingly, approximately 40% of the Nms are species-specific modifications that do not exist in yeast, humans, or plants, and 40% of the species-specific predicted modifications are located in unique positions outside the highly conserved domains. Although most of the guide RNAs were found in reiterated clusters, a few single-copy genes were identified. The large repertoire of modifications and guide RNAs in trypanosomes suggests that these modifications possibly play a central role in these parasites.

Animals↗
PubMed2005

Single-nucleosome mapping of histone modifications in S. cerevisiae.

Covalent modification of histone proteins plays a role in virtually every process on eukaryotic DNA, from transcription to DNA repair. Many different residues can be covalently modified, and it has been suggested that these modifications occur in a great number of independent, meaningful combinations. Published low-resolution microarray studies on the combinatorial complexity of histone modification patterns suffer from confounding effects caused by the averaging of modification levels over multiple nucleosomes. To overcome this problem, we used a high-resolution tiled microarray with single-nucleosome resolution to investigate the occurrence of combinations of 12 histone modifications on thousands of nucleosomes in actively growing S. cerevisiae. We found that histone modifications do not occur independently; there are roughly two groups of co-occurring modifications. One group of lysine acetylations shows a sharply defined domain of two hypo-acetylated nucleosomes, adjacent to the transcriptional start site, whose occurrence does not correlate with transcription levels. The other group consists of modifications occurring in gradients through the coding regions of genes in a pattern associated with transcription. We found no evidence for a deterministic code of many discrete states, but instead we saw blended, continuous patterns that distinguish nucleosomes at one location (e.g., promoter nucleosomes) from those at another location (e.g., over the 3' ends of coding regions). These results are consistent with the idea of a simple, redundant histone code, in which multiple modifications share the same role.

Chromatin Immunoprecipitation↗
PubMed2026

Characterization of METTL3/14-mediated m6A modification in human transcriptome using Nanopore direct RNA sequencing.

Post-transcriptional RNA modifications modulate diverse aspects of RNA metabolism. N6-methyladenosine (m6A), one of the most abundant internal RNA modifications, is deposited by the core methyltransferase complex, METTL3 and METTL14. Oxford Nanopore Technologies (ONT) platform permits direct, single RNA molecule sequencing while preserving native modifications. However, without rigorous benchmarking, the accuracy and reproducibility of modification detection remain uncertain. Here, we leveraged ONT to comprehensively profile bona fide m6A modifications in cellular RNAs at single-nucleotide resolution by integrating two direct RNA sequencing chemistries (RNA002 and RNA004) with the m6Anet and Dorado modification-detection models. We independently depleted METTL3 and METTL14 in human cells and rigorously validated modification calls through several assays and independent orthogonal methods (GLORI and miCLIP). We find that Dorado detected a higher number of m6A events and enabled simultaneous detection of other RNA modifications (5-methylcytosine, pseudouridine, and inosine). Pairing Dorado with an in vitro transcribed, unmodified control under stringent filtering, we provide compelling evidence supporting a global reduction in m6A sites and stoichiometry within coding sequences and across genes, particularly in highly modified genes and sites, and at consensus DRACH motifs. We report a differential and complex regulation of modified transcripts, accompanied by a global reduction in poly(A) tail length. Notably, METTL3 and METTL14 depletion produced distinct transcript-specific effects, supporting non-redundant roles within the m6A writer complex. Together, our study illustrates a notable advancement of ONT capabilities and establishes a robust transcriptome-wide framework for RNA modification detection, thereby laying the groundwork for exploring the contribution of METTL3/METTL14 to cellular functions and disease.

Humans↗
PubMed2000

Signaling to p53: breaking the posttranslational modification code.

In unstressed cells, the tumor suppressor protein p53, a tetrameric transcription factor, is present in a latent state and is maintained at low levels through targeted degradation. A variety of cellular stresses including DNA damage, hypoxia, nucleotide depletion, viral infection, and cytokine-activated signaling pathways that transiently stabilize the p53 protein, cause it to accumulate in the nucleus, and activate it as a transcription factor. Activation leads either to growth arrest at the G1/S or G2/M transitions of the cell cycle or to apoptosis. The molecular mechanisms by which stabilization and activation occur are incompletely understood, but accumulating evidence points to roles for multiple posttranslational modifications in mediating these events through several potentially interacting but distinct pathways. Both the approximately 100 amino acid N-terminal and approximately 90 amino acid C-terminal domains are highly modified by phosphorylation and acetylation, whereas modifications to the central sequence-specific DNA binding domain have not been reported. Seven serines and one threonine in the first 46 residues of the transactivation domain and four to five serines in the carboxyl-terminal domain are now known to be phosphorylated, and Lys320 and Lys382 in the carboxyl-terminal domain (human p53) can be acetylated. Antibodies that recognize p53 only when it has been modified at specific sites have been developed by several laboratories, and studies with these have shown that most of the known posttranslational modifications are induced when cells are exposed to DNA-damaging agents. Exceptions are Ser378, which is reported to be constitutively phosphorylated, and Ser376, which is dephosphorylated in response to DNA damage. These recent results, coupled with biochemical and genetic studies, suggest that several amino-terminal phosphorylations can be important in stabilizing p53 in response to DNA damage and in directing acetylation at C-terminal sites. DNA damage-induced modifications to the C-terminus inhibit the ability of this domain to negatively regulate sequence-specific DNA binding either by inducing a conformational change in the protein or by inhibiting non-sequence-specific DNA binding by the C-terminus. C-terminal modifications also modulate the oligomerization state of p53, and may modulate nuclear import/export. Modifications in response to DNA damage to other components that interact with p53 may also be important. In most cases, clear roles for specific modifications, interactions among individual modifications, and the enzymes responsible for each modification remain to be defined. Nevertheless, the field appears poised for major advances in the understanding of the molecular mechanisms that regulate p53 function.

Acetylation↗
PubMed2000

Responsiveness of the state of O-linked N-acetylglucosamine modification of nuclear pore protein p62 to the extracellular glucose concentration.

O-linked N-acetylglucosamine (O-GlcNAc) modification has been described in many proteins, including nuclear pore glycoproteins. In the present study we investigated the effect of extracellular glucose on the level of modification of nuclear pore protein p62 by O-GlcNAc. We found that exposure of cells to a high concentration of glucose caused an increased level of modification of p62 with O-GlcNAc, whereas the modification of other proteins did not change detectably. The increased O-GlcNAc modification of p62 in response to glucose required the metabolism of glucose to glucosamine. The exposure of the cells to glucosamine itself also caused increased O-GlcNAc modification, whereas mannosamine and galactosamine did not. Furthermore, changes in glucose concentrations within the physiological range induced the O-GlcNAc modification in p62 in rat aortic smooth-muscle cells, indicating that this modification of p62 might occur in an intact animal. These results imply that the ambient glucose concentration has an impact on the level of O-GlcNAc modification of proteins such as p62, and that functional changes in some of these proteins might ensue.

Acetylglucosamine↗
PubMed2004

[Effect of chemical modification of grafts on the survival improvement post haploidentical bone marrow transplantation in mice].

OBJECTIVE: Human leukocyte antigen (HLA) haploidentical bone marrow is a potential source of donor to children for its availability. The drawback is deleterious graft versus host disease (GVHD) reaction post transplantation because of the incompatibility of HLA antigen expression between donors and recipients, in which donor T lymphocyte is stimulated to proliferate and differentiate. The methoxy polyethylene glycol (mPEG) is a kind of amphoteric compound without immunogenicity, which was used to modify various proteins covalently and to prepare the versatile blood type. If mPEG modification blocks the activation of T cells in grafts, GVHD reaction probably would become less serious and transplantation might become successful. The aim of this study was to verify the improvement of haploidentical bone marrow transplantation (BMT) in a murine model by using mPEG of certain concentration to modify the grafts. METHODS: Male BALB/c mice were chosen as the donor, and female CB(6)F(1) mice as the recipient. There were three groups of mPEG modification, non-modification and irradiation control, and 20 mice in each group. The modified and non-modified mixture of bone marrow and spleen cells (as T lymphocytes) were transplanted to haploidentical lethally irradiated CB(6)F(1) mice via the tail vein. After the transplant, the hematopoietic recovery, survival rate, acute graft versus host disease (aGVHD) and chromosomal karyotype were analyzed and compared with controls. RESULTS: Seventy-five percent (15/20) of mice survived in the group of mPEG modification, while only 40% (8/20) survived in the group without the modification (chi(2) = 5.01, P = 0.025). And 100% mice died in the group of the irradiation control within 2 weeks. The hematopoietic recovery in the group of mPEG modification was show n to be faster than that in the group without modification (P < 0.05). Histopathological examination of the skin, liver and intestine showed typical signs of aGVHD, but the GVHD grading in the group of modification was less severe. The recipient mice in both groups of transplantation surviving for more than 75 days showed complete donor-type implantation by the chimerism examination. CONCLUSION: The modification of grafts by mPEG could alleviate aGVHD and improve the survival rate of mice after the haploidentical bone marrow transplantation.

Animals↗
PubMed1985

Covalent modification of proteins by mixed-function oxidation: recognition by intracellular proteases.

Mixed-function oxidation of E. coli glutamine synthetase is a site-specific reaction involving covalent modification of specific amino acid residues. It causes loss of a specific histidine residue which is thought to be at one of the metal binding sites. The modified enzyme is catalytically inactive. Oxidative modification causes enhanced susceptibility to proteolytic attack and several different types of proteases recognize the oxidatively modified enzyme. This specific covalent modification increases the rate of degradation of glutamine synthetase to about the same extent as major structural modifications such as relaxation, subunit dissociation and denaturation. Moreover, the oxidative modification is one that is likely to occur in vivo. Adenylylation, which causes reversible inactivation of glutamine synthetase, has no effect on rate of proteolysis. We propose that the degradation of E. coli glutamine synthetase occurs by a two-step process. Control of the degradation is likely to be at the first step, which is inactivation by mixed-function oxidation of the enzyme. Metabolic control of the degradation process and the link with the nutritional state of the cell could be achieved by substrate protection against oxidative modification. At present, the apparent energy requirement of the degradation process is unexplained. Although most of our studies have involved E. coli glutamine synthetase, there is evidence that oxidative modification may be a general mechanism by which proteins are marked for degradation. Many enzymes are inactivated by oxidative modification which can be catalyzed by a variety of mixed-function oxidase systems. Several different types of intracellular proteases in E. coli and mammalian cells preferentially degrade the oxidized form of glutamine synthetase. Oxidative modification of proteins can occur in vivo and may be involved in intracellular protein turnover. It has also been implicated in host defense mechanisms and in aging.

Amino Acid Sequence↗
PubMedDate not supplied

[Pyruvate dehydrogenase from pigeon breast muscle. Chemical modification of the enzyme associated with conformation changes in the protein molecule].

The two-phase character of essential histidine residues modification of pyruvate dehydrogenase component of pyruvate dehydrogenase complex from pigeon breast muscle by diethylpyrocarbonate has been demonstrated. The relative amplitude of the fast phase increases with increasing the modificator concentration. The model of chemical modification of dimeric enzyme where the modification of the residue in one subunit leads to the change of reactivity of corresponding residue in the other subunit is used for the description of inactivation kinetics. The expression for the diminishing of enzyme activity in the course of chemical modification and the methods of kinetic parameters estimation have been proposed. The following values of kinetic parameters for the modification of pyruvate dehydrogenase component by diethylpyrocarbonate were obtained (pH 6.0; 20 degrees C): k1 = 6400 +/- 400 M-1 min-1 (the microscopic rate constant for the modification of histidine residue in the intact dimer), k2 = 890 +/- 200 M-1 min-1 (the rate constant for the modification of histidine residue in the intact subunit in the dimer which contains one modified subunit) and kt = 0.9 +/- 0.2 min-1 (the rate constant for conformational transition of the dimer induced by modification of histidine residue in one of the subunits in the dimeric molecule).

Animals↗
PubMed1987

Biosynthesis and secretion of the rat core-specific lectin. Relationship of post-translational modification and assembly to attainment of carbohydrate binding activity.

A soluble lectin, the core-specific lectin (CSL), is synthesized and secreted by rat hepatocytes and the rat hepatoma cell line, H-4-II-E. This lectin binds mannose and N-acetylglucosamine residues in the "core" region of Asn-linked oligosaccharides. Secretion of the CSL was found to occur over an extended period of time, greater than 4 h being required for secretion of 50% of the lectin (Brownell, M. D., Colley, K. J., and Baenziger, J. U. (1984) J. Biol. Chem. 259, 3925-3932). We have determined that following synthesis in the endoplasmic reticulum, the CSL is rapidly transported to the Golgi where it is retained for an extended period of time prior to secretion. The lectin undergoes two post-translational modifications within the Golgi: an increase from Mr 24,000 to 25,000 and a progressive decrease in pI with an accompanying increase in Mr to a final value of 26,000. The lectin is also assembled into high molecular weight complexes of 150-260 X 10(3) and acquires the ability to bind carbohydrate in the Golgi. In hepatoma cells, the 24,000-25,000 modification is completed 20 min after initiation of synthesis. Assembly of the CSL subunits into high molecular weight complexes, acquisition of carbohydrate binding activity, and the 25,000-26,000 modification occur between 20 and 80 min after initiation of synthesis. These events have slower kinetics in primary hepatocytes and this allowed us to determine that the sequence of these biosynthetic events is: the 24,000-25,000 modification, complex assembly, the 25,000-26,000 modification, and acquisition of carbohydrate binding activity. The 24,000-25,000 modification occurs prior to complex assembly. Complex assembly may occur prior to, or concomitant with, the 25,000-26,000 modification. Assembly into the oligomeric form and the 25,000-26,000 modification correlate with the attainment of carbohydrate binding activity. The kinetics of CSL modification and assembly cannot account for its retention within the Golgi. Interaction with Golgi components either through carbohydrate binding or another interaction, may act to selectively retain the lectin within the Golgi.

Animals↗
PubMed1998

Surface modification of polyurethane heart valves: effects on fatigue life and calcification.

Polyurethane heart valves can be functionally durable with minimal calcification, in vitro. In vivo, these characteristics will depend on the resistance of the polyurethane to thrombogenesis and biodegradation. Surface modification may improve the polyurethane in these respects, but may adversely affect calcification and durability. This study investigates the effects of surface modifications of two polyurethane heart valves (PEU and PEUE) on their in vitro fatigue and calcification behaviour. Modifications included heparin, taurine, 3-aminopropyltriethoxysilane and polyethylene oxide (PEO). Neither hydrodynamic function nor leaflet thickness distribution was significantly altered by surface modification. PEO-modification was detrimental to valve fatigue durability and calcification. Heparin, taurine or aminosilane modifications of PEU valves increased durability. Aminosilane modification of PEUE valves increased durability compared with PEO modification. Appropriate surface modification may be useful to improve blood compatibility of implantable polyurethanes, and may also be advantageous as regards fatigue durability of flexing materials in longterm applications.

Analysis of Variance↗
PubMed1998

Cloning and expression of the ApaLI, NspI, NspHI, SacI, ScaI, and SapI restriction-modification systems in Escherichia coli.

The genes encoding the ApaLI (5'-GTGCAC-3'), NspI (5'-RCATGY-3'), NspHI (5'-RCATGY-3'), SacI (5'-GAGCTC-3'), SapI (5'-GCTCTTCN1-3', 5'-N4GAAGAGC-3') and ScaI (5'-AGTACT-3') restriction-modification systems have been cloned in E. coli. Amino acid sequence comparison of M.ApaLI, M.NspI, M.NspHI, and M.SacI with known methylases indicated that they contain the ten conserved motifs characteristic of C5 cytosine methylases. NspI and NspHI restriction-modification systems are highly homologous in amino acid sequence. The C-termini of the NspI and NlaIII (5'-CATG-3') restriction endonucleases share significant similarity. 5mC modification of the internal C in a SacI site renders it resistant to SacI digestion. External 5mC modification of a SacI site has no effect on SacI digestion. N4mC modification of the second base in the sequence 5'-GCTCTTC-3' blocks SapI digestion. N4mC modification of the other cytosines in the SapI site does not affect SapI digestion. N4mC modification of ScaI site blocks ScaI digetion. A DNA invertase homolog was found adjacent to the ApaLI restriction-modification system. A DNA transposase subunit homolog was found upstream of the SapI restriction endonuclease gene.

Amino Acid Sequence↗
PubMed2025

Proteins driving liquid-liquid phase separation and histone modifications cooperatively associate with chromatin looping and transcriptional regulation.

BACKGROUND: Although liquid-liquid phase separation (LLPS) proteins are known to participate in genome organization and transcriptional regulation through the formation of biomolecular condensates, their functional interplay with other regulatory proteins and histone modifications in chromatin loop formation remains poorly characterized. By combining Hi-C chromatin interaction data with ChIP-seq profiles of 12, 27, and 24 LLPS proteins in GM12878, K562, and HepG2 cell lines, respectively, we identified chromatin loops associated with LLPS proteins and systematically analysed patterns of cooperative protein binding and histone modification enrichment within these loop-associated peaks. RESULTS: We identified 162, 313, and 431 chromatin loops associated with LLPS proteins in GM12878, K562, and HepG2 cell lines, respectively. These loops were relatively small in size and predominantly anchored at enhancer regions. Examination of cooperative binding of proteins within loop-associated peaks revealed that transcriptional repressor IKZF1, HDAC1, and SAP130 most frequently co-localized with LLPS proteins in GM12878, K562, and HepG2 cells, respectively. Further analysis of histone modification enrichment patterns revealed that active histone modifications, such as H3K4me2, H3K4me3, H3K9ac, and H3K27ac, co-localized at loop-associated peaks, with H3K4me1 exhibiting additional specific co-localization with these four histone modifications at enhancer-localized loop-associated peaks. Notably, bivalent chromatin domains where H3K27me3 co-localized with active histone modifications were identified at promoter-localized loop-associated peaks in HepG2 cells, and elevated H3K27me3 occupancy at these peaks was associated with transcriptional repression of target genes. Moreover, quantitative RNA-seq analysis revealed that the expression of target genes associated with enhancer-promoter loops was correlated with both the binding of LLPS proteins and the enrichment patterns of histone modifications within their ChIP-seq peaks at loop anchors. CONCLUSIONS: Our study suggests that LLPS proteins may cooperate with transcriptional repressors to facilitate chromatin looping. Furthermore, local enrichment of histone modifications at loop-associated peaks provides additional regulatory control over chromatin architecture and gene transcription.

Humans↗
PubMed2001

Post-translational modifications of proteins: acetylcholinesterase as a model system.

Analysis of the expressed protein complement of cells requires knowledge of the diversity of post-translational modifications that can occur and which can be transient or permanent. The modifications range from amino acid changes through to the addition of macromolecules: lipid, carbohydrate or protein. Many variants of the common amino acids can occur, which can affect the structure or function of the protein. The major class of modification, however, is represented by glycosylation, N-linked, O-linked, or glycosylphosphatidylinositol(GPI)-linked. Such modifications have roles in protein stability and folding, targeting and recognition. Glycosylated proteins can be found in all cellular compartments and, intracellularly, O-GlcNAc modification is commonplace. Lipid modification of proteins (acylation, prenylation, GPI-anchoring) is also common, resulting in membrane association, and can play an important role in cell signalling. Targeting and turnover of proteins can also be mediated via covalent protein addition, for example by members of the ubiquitin family. Limited proteolysis as a post-translational modification will be discussed, focusing on the family of membrane protein secretases, in particular in relation to the Alzheimer's amyloid precursor protein. Finally, acetylcholinesterase will be used as a model example to illustrate the diversity of modifications occurring on a single protein.

Acetylcholinesterase↗
PubMed2007

The effect of chemical modification of amino acid side-chains on collagen degradation by enzymes.

In this study, the effects of specific chemical modifications of amino acid side-chains on the in vitro enzyme degradation of type I collagen was studied. Two monofunctional epoxides of different size and chemistry were used to modify lysine and methylglyoxal was used to modify arginine. Lysine residues were modified using glycidol, a small hydrophilic reagent or n-butylglycidylether, a larger hydrophobic reagent. Amino acid analysis, swelling measurements, in vitro enzyme degradation analyses (using either collagenase, trypsin, acetyltrypsin, or cathepsin B), and gel chromatography were used to determine the effects of each chemical modification on purified type I collagen. Collagen solubilization by enzymes depended upon the size and chemistry of epoxides used to modify lysine residues. Modification of lysine residues by glycidol and arginine modification by methylglyoxal together significantly reduced collagen solubilization by acetyltrypsin and collagenase, whereas increased collagen solubilization was observed for all enzymes after lysine modification with n-butylglycidylether combined with arginine modification by methylglyoxal. Gel chromatographic analyses of collagen fragments solubilized by acetyltrypsin from type I collagen revealed that both the extent of solubilization and sites of cleavage were altered after lysine and arginine modification. In contrast, lysine and arginine modification only altered the amount of collagen solubilized by collagenase and had no effect on the amount collagen solubilized by cathepsin B. The ability to modulate the enzyme degradation of collagen-based materials as demonstrated in this study may facilitate the design of novel scaffolds for tissue regeneration or collagen-based drug/protein/gene delivery systems.

Amino Acids↗
PubMed2006

Phosphorylation and glycosylation interplay: protein modifications at hydroxy amino acids and prediction of signaling functions of the human beta3 integrin family.

Protein functions are determined by their three-dimensional structures and the folded 3-D structure is in turn governed by the primary structure and post-translational modifications the protein undergoes during synthesis and transport. Defining protein functions in vivo in the cellular and extracellular environments is made very difficult in the presence of other molecules. However, the modifications taking place during and after protein folding are determined by the modification potential of amino acids and not by the primary structure or sequence. These post-translational modifications, like phosphorylation and O-linked N-acetylglucosamine (O-GlcNAc) modifications, are dynamic and result in temporary conformational changes that regulate many functions of the protein. Computer-assisted studies can help determining protein functions by assessing the modification potentials of a given protein. Integrins are important membrane receptors involved in bi-directional (outside-in and inside-out) signaling events. The beta3 integrin family, including, alpha(IIb)beta3 and alpha(v)beta3, has been studied for its role in platelet aggregation during clot formation and clot retraction based on hydroxyl group modification by phosphate and GlcNAc on Ser, Thr, or Tyr and their interplay on Ser and Thr in the cytoplasmic domain of the beta3 subunit. An antagonistic role of phosphate and GlcNAc interplay at Thr758 for controlling both inside-out and outside-in signaling events is proposed. Additionally, interplay of GlcNAc and phosphate at Ser752 has been proposed to control activation and inactivation of integrin-associated Src kinases. This study describes the multifunctional behavior of integrins based on their modification potential at hydroxyl groups of amino acids as a source of interplay.

Amino Acid Sequence↗
PubMed2004

The RESID Database of Protein Modifications as a resource and annotation tool.

The RESID Database of Protein Modifications is a comprehensive collection of annotations and structures for protein modifications and cross-links including pre-, co-, and post-translational modifications. The database provides: systematic and alternate names, atomic formulas and masses, enzymatic activities that generate the modifications, keywords, literature citations, Gene Ontology (GO) cross-references, protein sequence database feature table annotations, structure diagrams, and molecular models. This database is freely accessible on the Internet through resources provided by the European Bioinformatics Institute (http://www.ebi.ac.uk/RESID), and by the National Cancer Institute--Frederick Advanced Biomedical Computing Center (http://www.ncifcrf.gov/RESID). Each RESID Database entry presents a chemically unique modification and shows how that modification is currently annotated in the protein sequence databases, Swiss-Prot and the Protein Information Resource (PIR). The RESID Database provides a table of corresponding equivalent feature annotations that is used in the UniProt project, an international effort to combine the resources of the Swiss-Prot, TrEMBL and PIR. As an annotation tool, the RESID Database is used in standardizing and enhancing modification descriptions in the feature tables of Swiss-Prot entries. As an Internet resource, the RESID Database assists researchers in high-throughput proteomics to search monoisotopic masses and mass differences and identify known and predicted protein modifications.

Databases, Factual↗
PubMed1999

High-throughput mass spectrometric discovery of protein post-translational modifications.

The availability of genome sequences, affordable mass spectrometers and high-resolution two-dimensional gels has made possible the identification of hundreds of proteins from many organisms by peptide mass fingerprinting. However, little attention has been paid to how information generated by these means can be utilised for detailed protein characterisation. Here we present an approach for the systematic characterisation of proteins using mass spectrometry and a software tool FindMod. This tool, available on the internet at http://www.expasy.ch/sprot/findmod.html , examines peptide mass fingerprinting data for mass differences between empirical and theoretical peptides. Where mass differences correspond to a post-translational modification, intelligent rules are applied to predict the amino acids in the peptide, if any, that might carry the modification. FindMod rules were constructed by examining 5153 incidences of post-translational modifications documented in the SWISS-PROT database, and for the 22 post-translational modifications currently considered (acetylation, amidation, biotinylation, C-mannosylation, deamidation, flavinylation, farnesylation, formylation, geranyl-geranylation, gamma-carboxyglutamic acids, hydroxylation, lipoylation, methylation, myristoylation, N -acyl diglyceride (tripalmitate), O-GlcNAc, palmitoylation, phosphorylation, pyridoxal phosphate, phospho-pantetheine, pyrrolidone carboxylic acid, sulphation) a total of 29 different rules were made. These consider which amino acids can carry a modification, whether the modification occurs on N-terminal, C-terminal or internal amino acids, and the type of organisms on which the modification can be found. We illustrate the utility of the approach with proteins from 2-D gels of Escherichia coli and sheep wool, where post-translational modifications predicted by FindMod were confirmed by MALDI post-source decay peptide fragmentation. As the approach is amenable to automation, it presents a potentially large-scale means of protein characterisation in proteome projects.

Acetylation↗