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PubMed2006

Using phage display to select antibodies recognizing post-translational modifications independently of sequence context.

Many cellular activities are controlled by post-translational modifications, the study of which is hampered by the lack of specific reagents due in large part to their ubiquitous and non-immunogenic nature. Although antibodies against specifically modified sequences are relatively easy to obtain, it is extremely difficult to derive reagents recognizing post-translational modifications independently of the sequence context surrounding the modification. In this study, we examined the possibility of selecting such antibodies from large phage antibody libraries using sulfotyrosine as a test case. Sulfotyrosine is a post-translational modification important in many extracellular protein-protein interactions, including human immunodeficiency virus infection. After screening almost 8000 selected clones, we were able to isolate a single specific single chain Fv using two different selection strategies, one of which included elution with tyrosine sulfate. This antibody was able to recognize sulfotyrosine independently of its sequence context in test peptides and a number of different natural proteins. Antibody reactivity was lost by antigen treatment with sulfatase or preincubation with soluble tyrosine sulfate, indicating its specificity. The isolation of this antibody signals the potential of phage antibody libraries in the derivation of reagents specific for post-translational modifications, although the extensive screening required indicates that such antibodies are extremely rare.

Amino Acid Sequence↗
PubMed2005

Discovery of disease-induced post-translational modifications in cardiac contractile proteins.

Post-translational modifications to myofilament proteins are essential for the regulation of cardiac function in both normal and disease states. Recent developments in the field of proteomics have produced a variety of useful tools to study protein modifications. Current applications of proteomic technologies in the study of modifications to myofilament proteins are summarized. The separation, identification and characterization of myofilament modifications using gel electrophoresis and mass spectrometry approaches are discussed. Each method is illustrated and evaluated with selected examples, and several powerful emerging technologies are assessed.

Animals↗
PubMed1989

Post-translational modifications of poliovirus proteins.

The post-translational modifications of poliovirus proteins have been investigated by analysis of glycosylation, sulphation, phosphorylation and acylation of the proteins made in the infected HeLa cells. No glycosylation or sulphation of proteins specific for virus-infected cells was apparent. A number of changes in the pattern of phosphorylated proteins took place. The specific myristylation of the structural protein VP4 and its precursors was clearly apparent. Acylation of viral proteins with oleic or palmitic acid was not detected. Myristylation took place in the presence of the protease inhibitor ZnCl2, but not in the presence of inhibitors of translation, such as cycloneximide and anysomycin.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1994

Post-translational modifications of water-soluble human lens crystallins from young adults.

Post-translational modifications of the water-soluble human lens crystallins from young adult donors were identified and located using electrospray ionization mass spectrometric analysis of the intact proteins and fast atom bombardment mass spectrometry of enzymatic digests. Peptides corresponding to all of the sequences of alpha A-, alpha B-, and beta B2-crystallins were found, permitting the entire sequences to be searched for modifications. The major portions of these three crystallins were not modified. Modifications of alpha A-crystallin that were detected included 2 phosphorylated Ser residues (1 of which appears to be unique to human lenses), deamidation at some Gln and Asn residues, a disulfide bond between Cys-131 and Cys-142, and loss of the COOH-terminal Ser residue. Three phosphorylated Ser residues, but no deamidation, were found in alpha B-crystallin. The molecular weights of neither the intact protein nor the peptides in the enzymatic digests indicated any post-translational modification of the principal beta-crystallin, beta B2. The molecular weights of the other beta- and gamma-crystallins for which sequences have been published suggested the presence of post-translational modifications or errors in the published sequences. Although enough peptides were found to establish the presence of specific proteins, peptides corresponding to all portions of these proteins were not found, and elucidation of these structures is not yet complete. This mass spectrometric characterization of the total water-soluble proteins from normal young adult lenses provides a reference data base for future investigations of the modifications present in aged and cataractous lenses.

Adult↗
PubMed1996

Hysteretic behavior and differential apparent stability properties of microtubule species emerge from the regulation of post-translational modifications of microtubules.

At the epigenetic level, microtubule diversity is generated by several mechanisms of reversible post-translational modifications of tubulin subunits. In most cases, modification enzymes preferentially act on the tubulin subunits of microtubules, whereas the substrate of the enzymes which ensure the reverse reaction is preferentially the alpha beta-dimer of nonpolymerized tubulin. Most modifications identified to date appear to be nearly ubiquitous within the animal kingdom. Moreover, modifications are generally not mutually exclusive, so that cellular microtubules often bear several distinct biochemical alterations whose biological role is yet unknown. Post-translational modifications often (but not always) occur on microtubule species with low turnover rate. However, in vitro comparison of the polymerization and depolymerization rates of modified or unmodified forms of tubulin did not reveal any significant difference between molecular species. Thus, post-translational modifications are thought to be the result rather than the cause of microtubule stability. We re-examine this contention in the light of a regulated kinetic scheme for multiple and non-exclusive enzymatic modifications of microtubules. This study shows that different apparent stability properties of microtubule species emerge under such a kinetic regulation, although all the species were assumed to have identical intrinsic stability properties. This model can be used to reinterpret the results of well-known studies bearing on the relationship between microtubule stability and post-translational modifications. Another important finding is that the existence of a regulation loop in one of the multiple pathways of enzymatic differentiation of microtubules endows the system with hysteretic properties. These properties may be viewed, under restrictive conditions, as a buffering mechanism for the transitions between microtubule growing and shrinking phases during fluctuations in the regulation of centrosomal nucleating activity.

Enzymes↗
PubMed1993

O-linked fucose and other post-translational modifications unique to EGF modules.

Three types of unusual post-translational modification have been found within conserved amino acid sequences in epidermal growth factor homology regions (EGF modules) of some multidomain proteins. beta-Hydroxyaspartate and beta-hydroxyasparagine are found within -Cys-Xxx-Asp/Asn-Xxx-Xxx-Xxx-Xxx-Tyr/Phe-Xxx-Cys-Xxx-Cys- sequences. (Xyl alpha 1-->3)Xyl alpha 1-->3Glc beta 1-->O-Ser glycans at conserved sites within -Cys-Xxx-Ser-Xxx-Pro-Cys- sequences have been reported in several proteins. Fuc alpha 1-->O-Thr/Ser modifications have been found at conserved sites within -Cys-Xxx-Xxx-Gly-Gly-Thr/Ser-Cys- sequences. More recently, it has been discovered that the Ser residue corresponding to the potential O-fucosylation site in human factor IX carries the novel tetrasaccharide NeuAc alpha 2-->6Gal beta 1-->4GlcNAc beta 1-->3Fuc alpha 1-->O-Ser; this tetrasaccharide can be considered to be an extension of the Fuc alpha 1-->O moiety. The consensus sequences for these post-translational modifications are in close proximity to each other; e.g. human factor IX has all three unusual modifications within a 12 amino acid linear sequence. In proteins with multiple EGF modules, the O-glycosidic modifications have been found only within the N-terminal EGF module; beta-hydroxyaspartate/asparagine residues are not restricted in the same fashion. Little is known yet about the functions of, or possible relationships between, any of these modifications.

Amino Acid Sequence↗
PubMed2006

Organismal differences in post-translational modifications in histones H3 and H4.

Post-translational modifications (PTMs) of histones play an important role in many cellular processes, notably gene regulation. Using a combination of mass spectrometric and immunobiochemical approaches, we show that the PTM profile of histone H3 differs significantly among the various model organisms examined. Unicellular eukaryotes, such as Saccharomyces cerevisiae (yeast) and Tetrahymena thermophila (Tet), for example, contain more activation than silencing marks as compared with mammalian cells (mouse and human), which are generally enriched in PTMs more often associated with gene silencing. Close examination reveals that many of the better-known modified lysines (Lys) can be either methylated or acetylated and that the overall modification patterns become more complex from unicellular eukaryotes to mammals. Additionally, novel species-specific H3 PTMs from wild-type asynchronously grown cells are also detected by mass spectrometry. Our results suggest that some PTMs are more conserved than previously thought, including H3K9me1 and H4K20me2 in yeast and H3K27me1, -me2, and -me3 in Tet. On histone H4, methylation at Lys-20 showed a similar pattern as H3 methylation at Lys-9, with mammals containing more methylation than the unicellular organisms. Additionally, modification profiles of H4 acetylation were very similar among the organisms examined.

Acetylation↗
PubMed2000

Subcellular differences in post-translational modification of barley 14-3-3 proteins.

Expression and post-translational modification of barley 14-3-3 isoforms, 14-3-3A, 14-3-3B and 14-3-3C, were investigated using isoform-specific antibodies. Although all three isoforms were shown to be present in the cytosolic, the nuclear and the microsomal cell fractions, differences in post-translational modification were identified for the different cell fractions. Germination-related modifications of 14-3-3 proteins were observed in the cytosol and the microsomal fraction, but not in the nucleus. In vitro proteolytic cleavage of 14-3-3 proteins using trypsin suggests that for 14-3-3A this change was caused by proteolytic cleavage of the unconserved C-terminal region.

14-3-3 Proteins↗
PubMed2005

Histone H4 post-translational modifications in chordate mitotic and endoreduplicative cell cycles.

Histone post-translational modifications mark distinct structural and functional chromatin states but little is known of their involvement in the progression of different cell cycle types across phylogeny. We compared temporal and spatial dynamics of histone H4 post-translational modifications during both mitotic and endoreduplicative cycles of the urochordate, Oikopleura dioica, and proliferating mammalian cells. Endocycling cells showed no signs of chromosome condensation or entry into mitosis. They exhibited an evolution of replication patterns indicative of reduced chromatin compartmentalization relative to proliferating mammalian cells. In the latter cells, published cell cycle profiles of histone H4 acetylated at lysine 16 (H4AcK16) or dimethylated at lysine 20 (H4Me2K20) are disputed. Our results, using different, widely used H4AcK16 antibodies, revealed significant antibody-specific discrepancies in spatial and temporal cell cycle regulation of this modification, with repercussions for interpretation of previous immunofluorescence and immunoprecipitation data based on these reagents. On the other hand, three different antibodies to H4Me2K20 revealed similar cell cycle profiles of this modification that were conserved throughout the mitotic cell cycle in urochordate and mammalian cells, with accumulation at mitosis and a decrease during S-phase. H4Me2K20 also cycled in endocycles, indicating that dynamics of this modification are not strictly constrained by the mitotic phase of the cell cycle and suggesting additional roles during G- and S-phase progression. This article contains Supplementary Material available at http://www.mrw.interscience.wiley.com/suppmat/0730-2312/suppmat/2005/95/spada.html.

Acetylation↗
PubMed2003

Thermal stability and aggregation of sulfolobus solfataricus beta-glycosidase are dependent upon the N-epsilon-methylation of specific lysyl residues: critical role of in vivo post-translational modifications.

Methylation in vivo is a post-translational modification observed in several organisms belonging to eucarya, bacteria, and archaea. Although important implications of this modification have been demonstrated in several eucaryotes, its biological role in hyperthermophilic archaea is far from being understood. The aim of this work is to clarify some effects of methylation on the properties of beta-glycosidase from Sulfolobus solfataricus, by a structural comparison between the native, methylated protein and its unmethylated counterpart, recombinantly expressed in Escherichia coli. Analysis by Fourier transform infrared spectroscopy indicated similar secondary structure contents for the two forms of the protein. However, the study of temperature perturbation by Fourier transform infrared spectroscopy and turbidimetry evidenced denaturation and aggregation events more pronounced in recombinant than in native beta-glycosidase. Red Nile fluorescence analysis revealed significant differences of surface hydrophobicity between the two forms of the protein. Unlike the native enzyme, which dissociated into SDS-resistant dimers upon exposure to the detergent, the recombinant enzyme partially dissociated into monomers. By electrospray mapping, the methylation sites of the native protein were identified. A computational analysis of beta-glycosidase three-dimensional structure and comparisons with other proteins from S. solfataricus revealed analogies in the localization of methylation sites in terms of secondary structural elements and overall topology. These observations suggest a role for the methylation of lysyl residues, located in selected domains, in the thermal stabilization of beta-glycosidase from S. solfataricus.

Amino Acids↗
PubMed2003

Identification of novel histone post-translational modifications by peptide mass fingerprinting.

The extent and pattern of histone post-translational modifications is a key determinant dictating the structure of chromatin. We employed mass spectrometry to map the post-translational modifications present on mammalian core histones. Using accurate peptide mass fingerprinting on proteolytic digests of purified histones, we identified more than 20 novel sites of histone modification. One newly identified site of methylation, histone H4 lysine 59, maps to the surface of the nucleosome in close proximity to the site of the only identified histone core modification, histone H3 lysine 79. Consistent with the role of histone H3 lysine 79 methylation in the formation of silent chromatin structure, histone H4 lysine 59 is essential for transcriptional silencing at the yeast silent mating loci and telomeres.

Alanine↗
PubMed2006

Detecting oxidative post-translational modifications in proteins.

Oxidative stress induces various post-translational modifications (PTM); some are reversible in vivo via enzymatic catalysis. The present paper reviews specific procedures for the detection of oxidative PTM in proteins, most of them including electrophoresis. Main topics are carbonylated and glutathionylated proteins as well as modification of selected amino acids (Cys, Tyr, Met, Trp, Lys).

Oxidation-Reduction↗
PubMed2005

Post-translational modifications influence transcription factor activity: a view from the ETS superfamily.

Transcription factors provide nodes of information integration by serving as nuclear effectors of multiple signaling cascades, and thus elaborate layers of regulation, often involving post-translational modifications, modulating and coordinate activities. Such modifications can rapidly and reversibly regulate virtually all transcription factor functions, including subcellular localization, stability, interactions with cofactors, other post-translational modifications and transcriptional activities. Aside from analyses of the effects of serine/threonine phosphorylation, studies on post-translational modifications of transcription factors are only in the initial stages. In particular, the regulatory possibilities afforded by combinatorial usage of and competition between distinct modifications on an individual protein are immense, and with respect to large families of closely related transcription factors, offer the potential of conferring critical specificity. Here we will review the post-translational modifications known to regulate ETS transcriptional effectors and will discuss specific examples of how such modifications influence their activities to highlight emerging paradigms in transcriptional regulation.

Animals↗
PubMed2005

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

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

Histones↗
PubMed1990

Biotination of proteins in vivo. A post-translational modification to label, purify, and study proteins.

Post-translational modification of proteins with biotin provides the means to specifically label proteins in vivo and to purify proteins from crude cell lysates. The carboxyl-terminal protein segments modified by reaction with biotin ligase are strongly conserved in nature. We have demonstrated that the proteins encoded by translational gene fusions of a number of heterologous proteins to these carboxyl-terminal sequences become biotinated in vivo. The minimum size of the protein segment needed to allow biotination of fusion proteins is 75 amino acids. This biotination sequence, although of bacterial origin, functions in Saccharomyces cerevisiae as well as in Escherichia coli. Fusion proteins are readily labeled with [3H]biotin in vivo and the labeling is highly specific due to the scarcity (less than 5) of biotinated protein species. Biotinated fusion proteins can be readily purified in native form by binding to columns of monomeric avidin followed by elution with buffers containing biotin. Alternatively, proteins can be purified in a denatured form in presence of 1% sodium dodecyl sulfate or 8 M urea. Thus, this technology allows purification by affinity chromatography of any protein to which a biotination sequence can be attached. The ability to specifically label a protein in vivo should have utility in studies such as intracellular protein trafficking and cytoskeletonal dynamics.

Amino Acid Sequence↗
PubMed2005

Post-translational modifications of naturally processed MHC-binding epitopes.

A variety of different post-translational modifications of peptides displayed by class I and II MHC molecules have now been described. Some modifications promote the binding of peptides to MHC molecules, and might also influence the ability of the peptide to be produced by antigen processing pathways. In some instances, the antigen processing components themselves are actually responsible for generating post-translational modifications. Finally, evidence is accumulating that modifications can be altered as a consequence of inflammation, transformation, apoptosis and aging. This leads to altered repertories of MHC-associated peptides, which may be important in immune responses associated with autoimmune diseases, infection and cancer.

Animals↗
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↗