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PubMed1995

Carbonyl-related posttranslational modification of neurofilament protein in the neurofibrillary pathology of Alzheimer's disease.

We present the first evidence for carbonyl-related posttranslational modifications of neurofilaments in the neurofibrillary pathology of Alzheimer's disease (AD). Two distinct monoclonal antibodies that consistently labeled neurofibrillary tangles (NFTs), neuropil threads, and granulovacuolar degeneration in sections of AD tissue also labeled the neurofilaments within axons of the white matter following modification by reducing sugars, glutaraldehyde, formaldehyde, or malondialdehyde. The epitope recognized by these two antibodies shows a strict dependency for carbonyl modification of the neurofilament heavy subunit. The in vivo occurrence of this neurofilament modification in the neurofibrillary pathology of AD suggests that carbonyl modification is associated with a generalized cytoskeletal abnormality that may be critical in the pathogenesis of neurofibrillary pathology. Furthermore, the data presented here support the idea that extensive posttranslational modifications, including oxidative stress-type mechanisms, through the formation of cross-links, might account for the biochemical properties of NFTs and their resistance to degradation in vivo.

Alzheimer Disease↗
PubMed1999

Posttranslational modification of CLN3 protein and its possible functional implication.

The CLN3 gene associated with Batten disease and encoding a novel protein of a predicted 438 amino acids was cloned in 1995 by the International Batten Disease Consortium. The function of CLN3 protein remains unknown. Computer-based analysis predicted that CLN3 may contain several posttranslational modifications. Thus, to study the posttranslational modification of CLN3 protein, we have expressed a full-length CLN3 protein as a C-terminal fusion with green fluorescent protein of the jellyfish Aequerea victoria in a Chinese hamster ovary cell line. Previously, we have shown that CLN3 is a glycosylated protein from lysosomal compartment, and now, by using in vivo labeling with 32P, detection with anti-phosphoamino acid antibodies, and phosphoamino acid analysis, we demonstrate that CLN3 is a phosphorylated protein. We demonstrate that CLN3 protein does not undergo mannose 6-phosphate modification and that it is a membrane protein. Furthermore, we show that the level of CLN3 protein phosphorylation may be modulated by several protein kinases and phosphatases activators or inhibitors.

Alkaline Phosphatase↗
PubMed1989

Application of fast atom bombardment mass spectrometry to posttranslational modifications of neuropeptides.

FABMS is a powerful and sensitive analytical technique capable of providing structural information unattainable by standard methods of peptide analysis. Many posttranslational modifications are undetectable by other routine analytical methods. In addition, FABMS is capable of providing information regarding posttranslational modifications at levels of peptide comparable to those required for other methods of analysis (10-1000 pmol). FABMS has had the effect on protein structure analysis that structure determination of any neuropeptide might now be considered incomplete without some form of mass spectrometric analysis. Much of the recent explosive increase in the use of mass spectrometry for solving problems in peptide structure analysis can be traced to improvements in methods capable of producing molecular ions from nonvolatile species. With the development of these methods, it can be expected that refinements of existing methods and new ionization methods will continue to increase the mass range and sensitivity available for peptide structure determination. For a brief review of other mass spectrometric methods applicable to peptides, see Delgass and Cooks.

Amino Acid Sequence↗
PubMed2002

Posttranslational modification of human alphaA-crystallin: correlation with electrophoretic migration.

alphaA-crystallin is a major protein component of the human lens. It is known to undergo posttranslational modification. This study was done to further elucidate the temporal and spatial nature of these posttranslational modifications and to correlate the modified forms with electrophoretic migration. We dissected normal human lenses into concentric shells of fiber cells, separated the proteins by two-dimensional electrophoresis, and identified modified forms by mass spectrometry. We found that alphaA-crystallin migrated as a major spot and in over 20 additional protein spots. The extent of modification correlated with the age of the fiber cells and the depth within a lens. A correlation was also seen between these parameters and the concentration of modified forms that had full-length sequences but migrated at more acidic positions. These proteins were phosphorylated, acetylated, and/or deamidated. A few proteins migrated to a more basic position than the major form of alphaA-crystallin. The locations of several species that were truncated after C-terminal residues Ser172 and Ser162 were identified. Each of these species had intact N termini. The similarity of the C-terminal cleavage sites found in alphaA- and alphaB-crystallins was noted.

Amino Acid Sequence↗
PubMed2007

Protein tyrosine phosphorylation and reversible oxidation: two cross-talking posttranslation modifications.

In addition to protein phosphorylation, redox-dependent posttranslational modification of proteins is emerging as a key signaling system, conserved throughout evolution, and influencing many aspects of cellular homeostasis. Recent data have provided new insight about the interplay between phosphorylation- and redox-dependent signaling, and reactive oxygen species have been included among intracellular signal transducers of growth factor and extracellular matrix receptors. Both tyrosine phosphorylation and thiol oxidation are reversible and dynamic, and this review will particularly focus on the cross-talk between these posttranslational protein regulatory means. Although these modifications share their reversibility, their effects on enzymatic activity of protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs) may be even opposite. Indeed, while tyrosine phosphorylation is frequently correlated to enzyme activation, thiol oxidation leads to inactivation of PTPs and to superactivation of PTKs. Several papers describe that both these modifications occur during the same input, (i.e., cell proliferation and motility induced by numerous growth factors and cytokines). The review will discuss several aspects of phosphorylation\oxidation interplay, describing both convergent and divergent features of the integrated and coordinated function of PTPs and PTKs during signaling.

Animals↗
PubMed1991

Independent Regulatory Aspects and Posttranslational Modifications of Two beta-Amylases of Rye : Use of a Mutant Inbred Line.

We have examined the occurrence/disappearance, tissue location, and posttranslational modification of beta-amylase proteins in rye (Secale cereale L.) kernels at three physiological stages (development, maturity, germination) with a normal inbred line and a mutant line exhibiting a high but incomplete beta-amylase deficiency. This deficiency corresponds to a lack of accumulation of beta-amylase activity in the endosperm and does not affect the level of activity in the outer pericarp and green tissues as compared to the normal line. Two antigenically related but distinct beta-amylases (I and II) were detected in the normal line (II being the major constituent) and only one (I) in the mutant line. I and II display very similar electrophoretic polymorphism. In both lines, I appears to be ubiquitous, although it disappears from the outer pericarp during ripening. Antigen II was present only in the normal line and appears to be specific for the endosperm and perhaps for the maternal green tissues of the seed. Posttranslational modifications occurring during germination, which are mimicked by the action of papain, affect II but not I. The two groups of beta-amylases are discussed in relation to recent reports indicating the presence of two types of beta-amylase with different functions and gene loci in barley and wheat.

Journal Article↗
PubMed1997

The UL13 protein kinase and the infected cell type are determinants of posttranslational modification of ICP0.

The herpes simplex virus infected-cell protein 0 (ICP0) acts as a promiscuous transactivator of genes introduced into eukaryotic cells by transfection or infection. The protein is highly posttranslationally modified by phosphorylation and nucleotidylylation. We have examined the electrophoretic mobility and phosphorylation of ICP0 in Vero and rabbit skin cells infected with wild-type virus or viruses from which the UL13 gene (DeltaUL13) encoding a protein kinase or the alpha22/US1.5 genes (Deltaalpha22/DeltaUS1.5) encoding putative transcriptional factors has been deleted. We report the following: (i) The accumulation of ICP0 and the electrophoretic mobility of ICP0 were dependent on the nature of the infected cell type and the presence of UL13. ICP0 encoded by wild-type virus accumulated to maximum levels earlier in infected Vero cells and its electrophoretic mobility was slower than that made in rabbit skin cells. In both Vero and rabbit skin cells infected with the DeltaUL13 virus, the prevailing ICP0 form migrated faster than that accumulating in the corresponding cells infected with wild-type virus. (ii) The alteration in electrophoretic mobility of ICP0 made in cells infected with DeltaUL13 virus was due to the absence of the UL13 protein and not to failure of posttranslational modification of Deltaalpha22/DeltaUS1.5 proteins inasmuch as the mobility of ICP0 in cells infected with Deltaalpha22/DeltaUS1.5 virus could not be differentiated from that of wild-type infected cells. (iii) ICP0 is extensively phosphorylated in infected cells even in the absence of UL13 protein. ICP0 is, however, a substrate for the UL13 kinase inasmuch as ICP0 was phosphorylated in mixtures of immune complexes of ICP0 and UL13. Complexes containing ICP0 only or infected cell lysate proteins reacting with preimmune serum from the rabbit immunized with UL13 protein failed to phosphorylate ICP0. (iv) In the absence of UL13, ICP22 is overproduced-an imbalance attributed to UL13. Thus, ICP22 regulates both the utilization of splice acceptor sites and the longevity of ICP0 mRNA (K. L. Carter and B. Roizman, 1996, Proc. Natl. Acad. Sci. USA 93, 12535-12540); UL13 is involved in the posttranslational modification of ICP0 and is required for both posttranslational processing and control of abundance of ICP22.

Animals↗
PubMed1985

Collagen biosynthesis in systemic scleroderma: regulation of posttranslational modifications and synthesis of procollagen in cultured fibroblasts.

Activities of prolyl hydroxylase (PH), lysyl hydroxylase (LH), and the collagen glycosyltransferases and the extent of the posttranslational modification of lysine residues in newly synthesized collagen were studied in fibroblast cultures obtained from 9 scleroderma patients. The rate of procollagen synthesis had increased more than 3-fold in 3 scleroderma fibroblast lines, but had not changed to the same extent in the others, even though these did not differ from the "high-producers" histologically, clinically, or immunohistologically. The activities of PH and LH correlated significantly with the rate of procollagen synthesis in the same cell lines (p less than 0.001), but the glycosyltransferase activities were not elevated in the scleroderma fibroblasts. Further studies nevertheless indicated that the extent of the posttranslational modification of lysine residues had not significantly changed in the procollagen synthesized by any of the scleroderma fibroblasts investigated.

Adult↗
PubMed1997

Posttranslational modifications of alpha- and beta-tubulin in Giardia lamblia, an ancient eukaryote.

Tubulin of Giardia lamblia, a representative of the oldest eukaryotes, was screened for posttranslational modifications. Mass spectrometry of the carboxy-terminal peptides documents a large number of variants. Both alpha- and beta-tubulin show polyglycylation with up to 20 and 15 extra glycyl residues respectively. Minor variants show a low level of glutamylation without or with glycylation. The glutamylation-specific antibody GT335 detects alpha- and beta-tubulin in immunoblots. The terminal tyrosine is fully retained in alpha-tubulin, which is completely acetylated at Lys-40. Thus except for the detyrosination/tyrosination cycle all posttranslational modifications known for higher eukaryotes are already present in Giardia.

Acetylation↗
PubMed2001

Posttranslational modifications of microfibril associated glycoprotein-1 (MAGP-1).

Microfibril-associated glycoprotein-1 (MAGP-1) is a small molecular weight protein associated with extracellular matrix microfibrils. Biochemical studies have shown that MAGP-1 undergoes several posttranslational modifications that may influence its associations with other microfibrillar components. To identify the sites in the molecule where posttranslational modifications occur, we expressed MAGP-1 constructs containing various point mutations as well as front and back half truncations in CHO cells. Characterization of transiently expressed protein showed that MAGP-1 undergoes O-linked glycosylation and tyrosine sulfation at sites in its amino-terminal half. This region of the protein also served as a major amine acceptor site for transglutaminase and mediated self-assembly into high molecular weight multimers through a glutamine-rich sequence. Fine mapping of the modification sites through mutational analysis demonstrated that Gln20 is a major amine acceptor site for the transglutaminase reaction and confirmed that a canonical tyrosine sulfation consensus sequence is the site of MAGP-1 sulfation. Our results also show that O-glycosylation occurs at more than one site in the molecule.

Amino Acid Sequence↗
PubMed2002

Top-down mass spectrometry of a 29-kDa protein for characterization of any posttranslational modification to within one residue.

A mass difference between the measured molecular weight of a protein and that of its DNA-predicted sequence indicates sequence errors and/or posttranslational modifications. In the top-down mass spectrometry approach, the measured molecular ion is dissociated, and these fragment masses are matched against those predicted from the protein sequence to restrict the locations of the errors/modifications. The proportion of the ion's interresidue bonds that are cleaved determines the specificity of such locations; previously, ubiquitin (76 residues) was the largest for which all such bonds were dissociated. Now, cleavages are achieved for carbonic anhydrase at 250 of the 258 interresidue locations. Cleavages of three spectra would define posttranslational modifications at 235 residues to within one residue. For 24 of the 34 possible phosphorylation sites, the cleavages of one spectrum would delineate exactly all -PO(3)H substitutions. This result has been achieved with electron-capture dissociation by minimizing the further cleavage of primary product ions and by denaturing the tertiary noncovalent bonding of the molecular ions under a variety of conditions.

Amino Acid Sequence↗
PubMed1996

Collagen type I of rat cortical and trabecular bone differs in the extent of posttranslational modifications.

This study sought to evaluate whether the architecture of the matrix of cortical and trabecular bone is exactly the same. For this purpose we analyzed the extent of some posttranslational modifications of type I collagen, which is the major component of bone matrix. Ten female and 10 male 100-day-old rats were sacrificed and the content of hydroxylysine, glycosylated hydroxylysine, and pyridinium cross-links of collagen from cortical and trabecular bone was determined. The amount of each compound was expressed as a molar ratio with hydroxyproline. The collagen posttranslational modification pattern appears to be the same in both sexes but with a higher extent of differences in females compared with males. Comparing cortical and trabecular bone, the former contains a higher amount of hydroxylysine residues whereas in the latter, glycosylation of hydroxylysine is higher and pyridinium cross-link concentration is lower. Moreover, an inverse linear relationship between glycosylated hydroxylysine and pyridinium cross-links concentration was established, both for female (r = -0.455, P = 0.04) and male rats (r = -0.426; P = 0.06). This paper discusses what these findings may mean in functional terms.

Adrenal Cortex Hormones↗
PubMed2004

Posttranslational modifications of alpha-tubulin of Toxoplasma gondii.

The posttranslational modifications of alpha-tubulin of Toxoplasma gondii were characterized by antibodies and biochemical analysis of the carboxy-terminal peptide. Alpha-Tubulin is acetylated and glutamylated. Side chains with up to three glutamate residues are linked to Glu445 of T. gondii alpha-tubulin. The data suggest that the site of glutamylation on alpha-tubulin is conserved over a broad range of species.

Amino Acid Sequence↗
PubMed2005

Mass spectrometric analysis of agonist effects on posttranslational modifications of the beta-2 adrenoceptor in mammalian cells.

Posttranslational modifications (PTMs) of the beta-2 adrenoceptor (B2AR) play a fundamental role in receptor regulation by agonists. We have examined the effects of several agonists on net levels of B2AR palmitoylation and phosphorylation using epitope tagging in stably transfected human embryonal kidney (HEK) 293 cells, immunoaffinity purification, and mass spectrometry combined with the method of stable isotope labeling by amino acids in cell culture (SILAC). Palmitoylation of Cys341 was confirmed and did not change detectably after 30 min exposure of cells to saturating concentrations of dopamine, epinephrine, or isoproterenol. However, all of these agonists produced a marked increase in net phosphorylation. Phosphorylation of the third cytoplasmic loop was increased to a similar degree by all three agonists, whereas differences between agonists were observed in net phosphorylation of the carboxyl-terminal cytoplasmic domain (isoproterenol approximately epinephrine >> dopamine). Interestingly, agonist-induced phosphorylation of the carboxyl-terminal cytoplasmic domain was observed exclusively in a proximal portion (between residues 339-369). None of the agonists produced detectable phosphorylation in a distal portion of the cytoplasmic tail, which contains all sites of agonist-induced phosphorylation identified previously by in vitro reconstitution. These results provide insight to agonist-dependent regulation of the B2AR in intact cells, suggest the existence of significant differences in regulatory phosphorylation events occurring between in vitro and in vivo conditions, and outline a general analytical approach to investigate regulated PTM of receptors in mammalian cells.

Adrenergic beta-Agonists↗
PubMed1986

Posttranslational modification of calmodulin in rat brain and pituitary.

The posttranslational modification of calmodulin has been studied in six brain regions and the anterior pituitary. Carboxylmethylation, calmodulin converting enzyme, and calmodulin (lysine) N-methyltransferase activities were determined. Incubation of calmodulin with cytosolic extracts of these tissues in the presence of the methyl donor [methyl-3H]-S-adenosyl-L-methionine and identification of labeled proteins by gel electrophoresis and fluorography indicated that calmodulin is a substrate for protein carboxylmethyltransferase in all tissues tested. In hippocampus, caudate nucleus, cerebral cortex, and anterior pituitary, but not in cerebellum, superior colliculus, brainstem, or diencephalon, a second methylated protein was found when calmodulin was added to incubation mixtures. This protein was shown to be identical to the previously described product of calmodulin converting enzyme. Converted calmodulin was isolated by fast protein liquid chromatography and shown to be des(Lys)calmodulin, lacking the carboxy terminal lysine residue of calmodulin. The anterior pituitary had by far the highest levels of calmodulin converting enzyme; this enzyme, in turn, was identified as a cobalt-stimulated carboxylpeptidase B. In contrast to the regional differences in these parameters, the levels of calmodulin (lysine) N-methyltransferase did not differ greatly among brain regions, although regional differences in the activity of this enzyme were statistically significant.

Amino Acids↗
PubMed2001

A major posttranslational modification of ezrin takes place during epithelial differentiation in the early mouse embryo.

The preimplantation development of the mouse embryo leads to the formation of two populations of cells: the trophectoderm, which is a perfect epithelium, and the inner cell mass. The divergence between these two lineages is the result of asymmetric divisions, which can occur after blastomere polarization at compaction. The apical pole of microvilli is the only asymmetric feature maintained during mitosis and polarity is reestablished only in daughter cells that inherit all or a sufficient part of this pole. To analyze the role of ezrin in the formation and stabilization of the pole of microvilli, we isolated and cultured inner cell masses (ICM). These undifferentiated cells can differentiate very quickly into epithelial cells. After isolation of the ICMs, ezrin relocalizes at the cell cortex before the formation of microvilli. This redistribution occurs in the absence of protein synthesis. The formation of microvilli at the apical surface of the outer cells of ICM correlates with a major posttranslational modification of ezrin. We show here that this posttranslational modification is not controlled by a serine/threonine kinase but an O-glycosylation may partially contribute to it. These data suggest that ezrin has at least two roles during development. First, ezrin may be involved in the formation of microvilli because it localizes at the cell cortex before microvilli appear in ICMs. Second, ezrin may stabilize the pole of microvilli because it is modified posttranslationally when microvilli form.

Animals↗
PubMed2005

Stable isotope labeling with amino acids in cell culture (SILAC) for studying dynamics of protein abundance and posttranslational modifications.

Stable isotope labeling with amino acids in cell culture (SILAC) is a simple and straightforward approach for in vivo incorporation of a tag into proteins for relative quantitation by mass spectrometry. SILAC is a simple, yet powerful, method for investigating the dynamics of protein abundance and posttranslational modifications. Here, we provide detailed instructions for using this method to study protein complexes, protein-protein interactions, and the dynamics of protein abundance and posttranslational modifications. We expect that SILAC will become a routine technique because of its applicability to most areas of cell biology. We have also developed a Web site (http://www.silac.org) to provide researchers with updated information about this method and related resources.

Amino Acids↗
PubMed1988

Homology of lysosomal enzymes and related proteins: prediction of posttranslational modification sites including phosphorylation of mannose and potential epitopic and substrate binding sites in the alpha- and beta-subunits of hexosaminidases, alpha-glucosidase, and rabbit and human isomaltase.

Recently developed computer programs, including secondary structure and epitopic site predictions, have been used to align lysosomal proteins for maximum homology, based on conservative interchanges, and the aligned sequences have been searched for potential sites for posttranslational modification, glycosylation, and binding and catalysis of substrate. The homology and prediction of the posttranslational modification of the alpha- and beta-subunits of hexosaminidase is in good agreement with previous observations, and an explanation of the differing substrate specificities of the two subunits is advanced. We show that the striking homology between alpha-glucosidase and isomaltase is reflected in the apparent conservation of the active site in both enzymes. Nonhomologous regions have been examined in detail in a search for binding sites for glycogen and maltose, and two such sites have been tentatively identified. A highly redundant consensus sequence for the phosphorylation of mannose in lysosomal proteins, YXX(Y, W, or F), is suggested.

Amino Acid Sequence↗