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PubMed2004

In vivo posttranslational modifications of the high mobility group A1a proteins in breast cancer cells of differing metastatic potential.

The high mobility group (HMG) proteins are important modulators of chromatin structure and gene transcription. Overexpression of HMGA1 proteins in vivo induces neoplastic transformation and promotes a highly metastatic cellular phenotype. This study focuses on characterization of HMGA1a in vivo posttranslational modification (PTM) patterns found in a nonmetastatic and two metastatic lines of MCF-7 human breast cancer cells of differing tumorigenic potential. PTM types and the amino acids on which they occur were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Mass analysis was restricted to MALDI peaks having less than +/-150 parts per million (ppm) error, thereby holding our analysis to a more stringent criterion than previously published work with the HMG proteins. Validation of MALDI-TOF MS analysis was accomplished utilizing electrospray ionization tandem mass spectrometry (ESI MS/MS) and manual analysis of ion fragmentation spectra. Patterns and sites of PTMs identified in this study suggest that HMGA1a proteins, like the histones, exhibit a biochemical modification "code" that relates to cellular function. For example, both increased levels of acetylation and a previously unidentified dimethylation of both lysine and arginine residues were found on HMGA1a proteins from metastatic cells compared to proteins found in their nonmetastatic precursors. Additionally, the types of modification present on lysine-45 (e.g., unmodified, acetylation, or dimethylation) varied, depending on the metastatic potential of cells. These findings suggest that examination of the PTM patterns on HMGA1 proteins may provide valuable information concerning the physiological and phenotypic state of mammalian cells.

Amino Acid Sequence↗
PubMed1991

Posttranslational modifications within the HIV-1 envelope glycoprotein which restrict virus assembly and CD4-dependent infection.

Alterations in two highly conserved N-linked glycosylation sites within the gp120 envelope glycoprotein of human immunodeficiency virus type I (HIV-1) implicated in the phenotype of a noncytopathic HIV-1 variant were introduced independently and in combination into a cytopathic, infectious HIV-1 clone by site-specific mutagenesis. Neither mutation affected the synthesis of HIV-1 envelope glycoproteins. However, one of the mutations restricted the ability of HIV-1 envelope to localize on the cell membrane and thus markedly impaired virus assembly. The HIV-1 assembly defect could be overcome in trans if site-specific mutants were packaged in HeLa cells constitutively producing wild-type HIV-1 envelope glycoprotein. In addition to inefficient virus assembly, this mutation impaired the ability of the virus to infect CD4+ T cells, but did not affect CD4-independent infection of muscle cells. These results suggest additional functions of posttranslational modification in virus replication (i.e., envelope glycoprotein transport). Given that such modifications can restrict CD4-mediated uptake without affecting CD4-independent uptake, variations in posttranslational env processing between different HIV-1 genotypes may affect virus tropism in vivo.

Amino Acid Sequence↗
PubMed1996

Posttranslational modifications in the C-terminal tail of axonemal tubulin from sea urchin sperm.

After proteolytic digestion of sperm tubulin from sea urchin Paracentrotus lividus, C-terminal peptides were isolated by chromatographic separations. The peptides were analyzed by Edman degradation and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. About 70% of the isolated C-terminal peptides were unmodified. The remaining modified peptides have undergone a combination of numerous posttranslational modifications generating significant heterogeneity of sperm tubulin. alpha-Tubulin is modified by detyrosylation, release of the penultimate glutamate, polyglutamylation, and polyglycylation. Glycylation and glutamylation can coexist within one alpha-tubulin isoform. beta-Tubulin undergoes polyglycylation but was not observed to be polyglutamylated. The number of units posttranslationally added reaches 11 and 12 glycyl units on beta- and alpha-tubulin, respectively. This is different from the polyglycylation of axonemal tubulin in Paramecium cilia where up to 40 added glycyl units were observed both on alpha- and beta-tubulin.

Amino Acid Sequence↗
PubMed2006

Posttranslational modifications of collagen studied by off-line coupling of HPLC and CE.

A typical example of non-enzymatic change of collagen is glycation (the Maillard reaction, formation of advanced glycation end products) resulting from the reaction of sugars with the epsilon-amino group of lysine. Posttranslational non-enzymatic modifications of collagen by sugars were studied. Collagenous tissues were incubated as a test protein separately with both glucose and ribose. The collagen mixture was digested by bacterial collagenase and separated by reversed-phase HPLC (in a Jupiter Proteo 90 A column). The eluate from this HPLC separation was collected as seven fractions and consecutively analysed by CE in a bare fused silica capillary (57/50 cm x 75 mm id) using 100 mM sodium 1-heptanesulfonate in 100 mM phosphate buffer, pH 2.5 (NaH2PO4 adjusted to pH by phosphoric acid). The chromatographic and electromigration behaviour of individual peptides varied considerably. This off-line HPLC-CE coupling made it possible to discover minor changes in the structure of collagen caused by posttranslational modifications. A new HPLC-CE technique for peptide analysis was developed, and applied to the identification of posttranslational modifications in slowly metabolised test proteins.

Animals↗
PubMed1984

Possible posttranslational modification, and its genetic control, in flax genotroph isozymes.

Environmentally induced flax genotrophs L and S show heritable shifts in the relative mobilities of peroxidase, esterase, and acid phosphatase isozymes, plus a number of nonspecific glycoproteins. All L isozymes migrated faster than corresponding S isozymes in 10% acrylamide gels. Various aspects of these shifts are reviewed here; it is proposed that posttranslational modification, probably of the carbohydrate moieties of these glycoproteins, underlies the shifts. This proposal is discussed in relation to the switch model for genotroph induction.

Acid Phosphatase↗
PubMed2005

Phosphorylation of cytochromes P450: first discovery of a posttranslational modification of a drug-metabolizing enzyme.

Cytochromes P450 (CYP) are important components of xenobiotic-metabolizing monooxygenases (CYP-dependent monooxygenases). Their regulation by induction, most commonly by transcriptional activation, mediated by xenobiotics, normally substrates of the corresponding CYP, is well known and has been widely studied. Our team has discovered an additional important regulation of xenobiotic-metabolizing CYPs pertaining to posttranslational modification by phosphorylation. Individual CYPs are phosphorylated by different protein kinases, leading to CYP isoenzyme-selective changes in the metabolism of individual substrates and consequent drastic changes in the control of genotoxic metabolites. Best studied are the CYP phosphorylations by the cAMP-dependent protein kinase A. Most recently, we discovered that cAMP not only leads to drastic changes in the activity of individual CYPs, but also to drastic changes in the nuclear localization of the CYP-related transcription factor Ah receptor (AHR). The consequences are very different from those of AHR nuclear translocation mediated by the classical ligands (enzyme inducers such as dioxin) and are likely to represent the long-sought physiological function of the AHR, its persistent disturbance by long-lived ligands such as dioxin may well be the reason for its high toxicity.

Animals↗
PubMed2002

Osteopontin posttranslational modifications, possibly phosphorylation, are required for in vitro bone resorption but not osteoclast adhesion.

Osteopontin (OPN), a phosphorylated bone matrix glycoprotein, is an Arg-Gly-Asp (RGD)-containing protein that interacts with integrins and promotes in vitro attachment of a number of cell types, including osteoclasts. Gene knockout experiments support the idea that OPN is important in osteoclastic activity. We hypothesize that posttranslational modifications (PTMs) of OPN can influence its physiological function. Previous studies have suggested that phosphorylation of OPN and bone sialoprotein (BSP) is necessary for promoting osteoclast adhesion. However, no reports have explored the importance of phosphoserines and other PTMs in OPN-promoted bone resorption. To study this question, we determined the activities of different forms of OPN and BSP in three in vitro assays: attachment of osteoclasts; formation of actin rings; and bone resorption. For each assay, cells were incubated for 4-24 h, in the presence or absence of RGDS or RGES peptides, to test the involvement of integrin binding. In addition to OPN, activities of milk OPN (fully phosphorylated) and recombinant OPN (rOPN, no phosphate) were compared. We purified two forms of OPN (OPN-2 and OPN-5), which differ in the level of phosphorylation, and compared their activities. For comparison, the activities of BSP and recombinant BSP (rBSP) were determined. All forms of OPN, including rOPN, significantly increased attachment of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts. BSP and rBSP also promoted cell attachment. After 4 h of incubation, the proportion of cells with actin rings was increased with OPN, milk OPN, and BSP. In the presence of RGDS peptide, osteoclast retraction and the disruption of actin rings were observed, whereas no effect was seen with RGES. In the resorption assay, the number of pits and the total resorbed area per slice were increased in the presence of OPN, milk OPN, and BSP. As in other assays, the OPN enhancement of resorption was inhibited by RGDS, but not RGES, peptides. Significantly, rOPN and rBSP did not promote bone resorption. OPN-5 promoted resorption to a greater extent than OPN-2, and milk OPN significantly stimulated resorption to a greater extent than OPN. Our data suggest that: (1) the RGD sequence of OPN is essential in OPN-mediated cell attachment, actin ring formation, and bone resorption; and (2) some form of PTM, possibly phosphorylation, is necessary for in vitro osteoclastic bone resorption, but not for cell attachment and actin ring formation.

Actins↗
PubMed2000

Cubilin expression and posttranslational modification in the canine gastrointestinal tract.

Cubilin is an endocytic receptor of the apical brush border membrane that is essential for intrinsic factor-mediated cobalamin absorption in small intestine. However, cubilin is more highly expressed in kidney and yolk sac, and recent molecular characterization of the receptor has focused on these tissues. The aim of this investigation was to examine tissue-specific cubilin expression and posttranslational modifications with an emphasis on the gastrointestinal tract. Intrinsic factor-cobalamin binding activity, cubilin immunoreactivity, and cubilin mRNA levels were determined in multiple segments of canine gastrointestinal mucosa and other tissues. These aspects of cubilin expression varied in parallel, suggesting that the major determinant of regional cubilin expression in the gastrointestinal tract is modulation of cubilin mRNA. Cell fractionation indicated that ileal cubilin is not strongly membrane associated. An approximately 185-kDa brush border specific and two >400-kDa precursor forms of cubilin were identified. Asparagine-linked oligosaccharide modifications characterized by differential glycosidase digestion of affinity-purified cubilin from ileal mucosa and renal cortex differed, but ileal and renal intracellular cubilin comigrated on SDS-PAGE at approximately 400 kDa after oligosaccharide removal, thus reconciling previous conflicting size estimates of the cubilin polypeptide.

Animals↗
PubMed2006

Adiponectin multimerization is dependent on conserved lysines in the collagenous domain: evidence for regulation of multimerization by alterations in posttranslational modifications.

Adiponectin is a secreted, multimeric protein with insulin-sensitizing, antiatherogenic, and antiinflammatory properties. Serum adiponectin consists of trimer, hexamer, and larger high-molecular-weight (HMW) multimers, and these HMW multimers appear to be the more bioactive forms. Multimer composition of adiponectin appears to be regulated; however, the molecular mechanisms involved are unknown. We hypothesize that regulation of adiponectin multimerization and secretion occurs via changes in posttranslational modifications (PTMs). Although a structural role for intertrimer disulfide bonds in the formation of hexamers and HMW multimers is established, the role of other PTMs is unknown. PTMs identified in murine and bovine adiponectin include hydroxylation of multiple conserved proline and lysine residues and glycosylation of hydroxylysines. By mass spectrometry, we confirmed the presence of these PTMs in human adiponectin and identified three additional hydroxylations on Pro71, Pro76, and Pro95. We also investigated the role of the five modified lysines in multimer formation and secretion of recombinant human adiponectin expressed in mammalian cell lines. Mutation of modified lysines in the collagenous domain prevented formation of HMW multimers, whereas a pharmacological inhibitor of prolyl- and lysyl-hydroxylases, 2,2'-dipyridyl, inhibited formation of hexamers and HMW multimers. Bacterially expressed human adiponectin displayed a complete lack of differentially modified isoforms and failed to form bona fide trimers and larger multimers. Finally, glucose-induced increases in HMW multimer production from human adipose explants correlated with changes in the two-dimensional electrophoresis profile of adiponectin isoforms. Collectively, these data suggest that adiponectin multimer composition is affected by changes in PTM in response to physiological factors.

2,2'-Dipyridyl↗
PubMed2004

Posttranslational modification of therapeutic proteins in plants.

Plants have emerged as an alternative to current systems for the production of therapeutic proteins. The advantages of plants for the low-cost and large-scale production of safe and biologically active mammalian proteins have been documented recently. A major advantage of transgenic plants over production systems that are based on yeast or Escherichia coli is their ability to perform most of the posttranslational modifications (PTMs) that are required for the bioactivity and pharmacokinetics of recombinant therapeutic proteins. Furthermore, recent advances in the control of PTMs in transgenic plants have made it possible for plants to perform, at least to some extent, human-like modifications of recombinant proteins. Hence, plants have become a suitable alternative to animal cell factories for the production of therapeutic proteins.

Biological Factors↗
PubMed1990

Posttranslational modification of class III beta-tubulin.

The charge heterogeneity of class III beta-tubulin (beta III) during neural development was analyzed by high-resolution isoelectric focusing/two-dimensional polyacrylamide gel electrophoresis in combination with site-specific proteolytic digestion and immunological detection. The number of beta III isoforms (charge variants) gradually increases from one in embryonic brain to seven in adult brain. All of the charge heterogeneity is due to posttranslationally modified sites located within the extreme C-terminal region of the beta III polypeptide. One beta III isoform is present in testis, the only other tissue in which this isotype is expressed. The testis beta III isoform cofocuses with the earliest-appearing embryonic brain beta III charge variant. Our results indicate that the posttranslational modifications of beta III are developmentally regulated, occur at more than one site, and are neuron-specific. The location of these modifications within the extreme C-terminal domain suggests that their function is to modulate the interaction of tubulin with microtubule-associated proteins.

Aging↗
PubMed1992

Posttranslational modification and subcellular localization of the p12 capsid protein of herpes simplex virus type 1.

We have previously shown that the 12-kDa capsid protein (p12) of herpes simplex virus type 1 (HSV-1) is a gamma 2 (true late) gene product encoded by the UL35 open reading frame (D. S. McNabb and R. J. Courtney, J. Virol. 66:2653-2663, 1992). To extend the characterization of p12, we have investigated the posttranslational modifications and intracellular localization of the 12-kDa polypeptide. These studies have demonstrated that p12 is modified by phosphorylation at serine and threonine residues. In addition, analysis of p12 by acid-urea gel electrophoresis has indicated that the protein can be resolved into three components, designated p12a, p12b, and p12c. Using isotopic-labeling and alkaline phosphatase digestion experiments, we have determined that p12a and p12b are phosphorylated forms of the protein, and p12c is likely to represent the unphosphorylated polypeptide. The kinetics of phosphorylation was examined by pulse-chase radiolabeling, and these studies indicated that p12c can be completely converted into p12a and p12b following a 4-h chase. All three species of p12 were found to be associated with purified HSV-1 virions; however, p12b and p12c represented the most abundant forms of the protein within viral particles. We have also examined the intracellular localization of p12 by cell fractionation and indirect immunofluorescence techniques. These results indicated that p12 is predominantly localized in the nucleus of HSV-1-infected cells and appears to be restricted to specific regions within the nucleus.

Animals↗
PubMed1999

Posttranslational modifications of cardiac and skeletal muscle proteins by reactive oxygen species after burn injury in the rat.

OBJECTIVE: To determine the involvement of oxidative damage in muscle wasting after burn injury. SUMMARY BACKGROUND DATA: Burn injury damages tissue at the site of the burn and also affects peripheral tissue. There is evidence to suggest that reactive oxygen species may be generated in increased amounts after burn, and these may contribute to wound healing and to posttranslational modifications of tissue constituents distant from the wound site. METHODS: The oxidation of muscle proteins was assessed, using the dinitrophenylhydrazine assay for carbonyl content, in muscles of rats after a full-thickness skin scald burn covering 20% of the total body surface area, over a 6-week period. In this model, rats failed to incur normal body weight or muscle weight gain. RESULTS: Soleus, extensor digitorum longus, diaphragm, and heart ventricle proteins were oxidatively damaged after injury. The extent of tissue protein oxidation, however, differed depending on the time points studied. In general, higher levels of protein carbonyl group formation, an indicator of oxidative damage, were found to occur within 1 to 5 days after injury, and the oxidized protein content of the various tissues decreased during the later stages. Both sarcoplasmic and myofibrillar carbonyl-containing proteins accumulated in diaphragm 3 days after burn injury and were rapidly removed from the tissue during a 2-hour in vitro incubation. This coincided with increased proteolytic activity in diaphragm. CONCLUSIONS: These observations suggest that the loss of proteins modified by reactive oxygen species may contribute to the burn-induced protein wasting in respiratory and other muscles by a proteolytically driven mechanism.

Animals↗
PubMed2000

Multiple posttranslational modifications at distinct sites contribute to heterogeneity of the lipoprotein cytochrome bo(3).

The heme-copper cytochrome oxidase of Escherichia coli (cytochrome bo(3)) was tagged with oligohistidine at the C-terminus of the small noncatalytic subunit IV. After detergent solubilization, the enzyme was purified by a one-step procedure with immobilized metal affinity chromatography. Using different cytochrome bo(3) constructs as reference, the products were investigated by mass spectroscopical and immunological methods. Several posttranslational modifications of subunits II, III, and IV were observed: (1) N-terminal methionines of subunits III and IV are split off. (2) Fifty percent of subunit III polypeptides are acetylated, presumably at the N-terminal alanine. (3) Lipoprotein processing of subunit II involves cleavage of the signal peptide. (4) Maturation of subunit II [Ma, J., Katsonouri, A., and Gennis, R. B. (1997) Biochemistry 36, 11298-11303] alters the structure of the N-terminal cysteine by N-palmitoylation and S-glyceryldipalmitoylation. (5) A hexapeptide is split off from the C-terminus of subunit II. This happens subsequently to the N-terminal lipoprotein processing step and is dependent on the growth state of cells.

Amino Acid Sequence↗
PubMed1997

Posttranslational modifications of the 5'-AMP-activated protein kinase beta1 subunit.

The AMP-activated protein kinase (AMPK) consists of catalytic alpha and noncatalytic beta and gamma subunits and is responsible for acting as a metabolic sensor for AMP levels. There are multiple genes for each subunit and the rat liver AMPK alpha1 and alpha2 catalytic subunits are associated with beta1 and gamma1 noncatalytic subunits. We find that the isolated gamma1 subunit is N-terminally acetylated with no other posttranslational modification. The isolated beta1 subunit is N-terminally myristoylated. Transfection of COS cells with AMPK subunit cDNAs containing a nonmyristoylatable beta1 reduces, but does not eliminate, membrane binding of AMPK heterotrimer. The isolated beta1 subunit is partially phosphorylated at three sites, Ser24/25, Ser182, and Ser108. The Ser24/25 and Ser108 sites are substoichiometrically phosphorylated and can be autophosphorylated in vitro. The Ser-Pro site in the sequence LSSS182PPGP is stoichiometrically phosphorylated, and no additional phosphate is incorporated into this site with autophosphorylation. Based on labeling studies in transfected cells, we conclude that alpha1 Thr172 is a major, although not exclusive, site of both basal and stimulated alpha1 phosphorylation by an upstream AMPK kinase.

AMP-Activated Protein Kinases↗
PubMed1998

Posttranslational modifications affect the activity of the human monocyte chemotactic proteins MCP-1 and MCP-2: identification of MCP-2(6-76) as a natural chemokine inhibitor.

Chemokines are important mediators in infection and inflammation. The monocyte chemotactic proteins (MCPs) form a subclass of structurally related C-C chemokines. MCPs select specific target cells due to binding to a distinct set of chemokine receptors. Recombinant and synthetic MCP-1 variants have been shown to function as chemokine antagonists. In this study, posttranslationally modified immunoreactive MCP-1 and MCP-2 were isolated from mononuclear cells. Natural forms of MCP-1 and MCP-2 were biochemically identified by Edman degradation and mass spectrometry and functionally characterized in chemotaxis and Ca2+-mobilization assays. Glycosylated MCP-1 (12 and 13.5 kDa) was found to be two- to threefold less chemotactic for monocytes and THP-1 cells than nonglycosylated MCP-1 (10 kDa). Natural, NH2-terminally truncated MCP-1(5-76) and MCP-1(6-76) were practically devoid of bioactivity, whereas COOH-terminally processed MCP-1(1-69) fully retained its chemotactic and Ca2+-inducing capacity. The capability of naturally modified MCP-1 forms to desensitize the Ca2+ response induced by intact MCP-1 in THP-1 cells correlated with their agonistic potency. In contrast, naturally modified MCP-2(6-76) was devoid of activity, but could completely block the chemotactic effect of intact MCP-2 as well as that of MCP-1, MCP-3, and RANTES. Carboxyl-terminally processed MCP-2(1-74) did retain its chemotactic potency. Although comparable as a chemoattractant, natural intact MCP-2 was found to be 10-fold less potent than MCP-1 in inducing an intracellular Ca2+ increase. It can be concluded that under physiologic or pathologic conditions, posttranslational modification affects chemokine potency and that natural MCP-2(6-76) is a functional C-C chemokine inhibitor that might be useful as an inhibitor of inflammation.

Amino Acid Sequence↗
PubMed2000

Posttranslational modifications of the KI-67 protein coincide with two major checkpoints during mitosis.

Ki-67 is a nuclear protein present in all proliferating cells that are in the active part of the cell division, but not in resting cells. This feature is extensively used in tumor diagnostics to estimate the growth fraction of a given cell population. We now demonstrate that the spatial and temporal regulation of the Ki-67 protein during the cell cycle is associated with mitosis-specific phosphorylation. These posttranslational modifications of the Ki-67 protein are accompanied by a characteristic redistribution of the protein from the interior of the nucleus to the periphery of the condensed chromosomes and vice versa. Phosphorylation could be suppressed by activating cell-cycle checkpoints that control the entry into mitosis through the activity of the cyclin B/cdc2 complex. In vitro experiments confirm that the presence of the cdc2 kinase and its regulatory subunit cyclin B is required for the phosphorylation of the Ki-67 protein. We further demonstrated that the Ki-67 protein is a new member of the family of MPM-2 reactive phosphoproteins, which includes both structural and functional proteins that are necessary for the control and timing of mitosis. Phosphorylation and dephosphorylation of the Ki-67 protein are therefore controlled by key regulatory structures of the cell cycle and occur at two hallmark events within the cell cycle: the breakdown and the reorganization of the nucleus during mitosis.

Adenosine Triphosphate↗
PubMed1999

Modulation of phosphate uptake and amphotropic murine leukemia virus entry by posttranslational modifications of PIT-2.

PIT-2 is a type III sodium phosphate cotransporter and the receptor for amphotropic murine leukemia viruses. We have investigated the expression and the functions of a tagged version of PIT-2 in CHO cells. PIT-2 remained equally abundant at the cell surface within 6 h following variation of the phosphate supply. In contrast, the efficiency of phosphate uptake and retrovirus entry was inversely related to the extracellular phosphate concentration, indicating that PIT-2 activities are modulated by posttranslational modifications of cell surface molecules induced by phosphate. Conformational changes of PIT-2 contribute to both activities, as shown by the inhibitory effect of sulfhydryl reagents known as inhibitors of type II cotransporters. A physical association of PIT-2 with actin was demonstrated. Modifications of the actin network were induced by variations of the concentrations of extracellular phosphate, cytochalasin D, or lysophosphatidic acid. They revealed that the formation of actin stress fibers determines the cell surface distribution of PIT-2, the internalization of the receptor in response to virus binding, and the capacity to process retrovirus entry. Thus, the presence of PIT-2 at the cell surface is not sufficient to ensure phosphate transport and susceptibility to amphotropic retrovirus infection. Further activation of cell surface PIT-2 molecules is required for these functions.

Actins↗