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PubMed1994

Identification of Posttranslationally Modified 18-Kilodalton Protein from Rice as Eukaryotic Translation Initiation Factor 5A.

Using anther-derived rice (Oryza sativa L.) cell-suspension cultures, we have identified an 18-kD protein that is posttranslationally modified by spermidine and is influenced by endogenous polyamine levels. The posttranslationally modified residue has been identified as the unusual amino acid hypusine [N[epsilon]-(4-amino-2-hydroxybutyl)lysine] by reverse-phase high-performance liquid chromatography and gas chromatography-mass-spectrometry analyses. Differential labeling of the protein with labeled amines provided evidence that the butylamine moiety of spermidine is the immediate precursor of the hypusine residue in the protein. The eukaryotic translation initiation factor 5A (eIF-5A) is the only known mammalian protein that undergoes a similar posttranslational modification with hypusine. The purified 18-kD protein co-electrophoreses with human translational initiation factor eIF-5A in both isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gels. The purified protein from rice stimulated methionyl-puromycin synthesis in vitro, indicating its functional similarity to mammalian eIF-5A. The results presented provide evidence that the posttranslationally modified 18-kD protein from rice containing hypusine is eIF-5A and suggest the conservation of hypusine-containing translation initiation factor eIF-5A in eukaryotes.

Journal Article↗
PubMed1993

Cell cycle regulation of the yeast Cdc7 protein kinase by association with the Dbf4 protein.

Yeast Cdc7 protein kinase and Dbf4 protein are both required for the initiation of DNA replication at the G1/S phase boundary of the mitotic cell cycle. Cdc7 kinase function is stage-specific in the cell cycle, but total Cdc7 protein levels remained unchanged. Therefore, regulation of Cdc7 function appears to be the result of posttranslational modification. In this study, we have attempted to elucidate the mechanism responsible for achieving this specific execution point of Cdc7. Cdc7 kinase activity was shown to be maximal at the G1/S boundary by using either cultures synchronized with alpha factor or Cdc- mutants or with inhibitors of DNA synthesis or mitosis. Therefore, Cdc7 kinase is regulated by a posttranslational mechanism that ensures maximal Cdc7 activity at the G1/S boundary, which is consistent with Cdc7 function in the cell cycle. This cell cycle-dependent regulation could be the result of association with the Dbf4 protein. In this study, the Dbf4 protein was shown to be required for Cdc7 kinase activity in that Cdc7 kinase activity is thermolabile in vitro when extracts prepared from a temperature-sensitive dbf4 mutant grown under permissive conditions are used. In vitro reconstitution assays, in addition to employment of the two-hybrid system for protein-protein interactions, have demonstrated that the Cdc7 and Dbf4 proteins interact both in vitro and in vivo. A suppressor mutation, bob1-1, which can bypass deletion mutations in both cdc7 and dbf4 was isolated. However, the bob1-1 mutation cannot bypass all events in G1 phase because it fails to suppress temperature-sensitive cdc4 or cdc28 mutations. This indicates that the Cdc7 and Dbf4 proteins act at a common point in the cell cycle. Therefore, because of the common point of function for the two proteins and the fact that the Dbf4 protein is essential for Cdc7 function, we propose that Dbf4 may represent a cyclin-like molecule specific for the activation of Cdc7 kinase.

Cell Cycle↗
PubMed2004

The SapB morphogen is a lantibiotic-like peptide derived from the product of the developmental gene ramS in Streptomyces coelicolor.

SapB is a morphogenetic peptide that is important for aerial mycelium formation by the filamentous bacterium Streptomyces coelicolor. Production of SapB commences during aerial mycelium formation and depends on most of the genes known to be required for the morphogenesis of aerial hyphae. Furthermore, the application of purified SapB to mutants blocked in morphogenesis restores their capacity to form aerial hyphae. Here, we present evidence that SapB is a lantibiotic-like peptide that is derived by posttranslational modification from the product of a gene (ramS) in the four-gene ram operon, which is under the control of the regulatory gene ramR. We show that the product of another gene in the operon (ramC) contains a region that is similar to enzymes involved in the biosynthesis of lantibiotics, suggesting that it might be involved in the posttranslational processing of RamS. We conclude that SapB is derived from RamS through proteolytic cleavage and the introduction of four dehydroalanine residues and two lanthionine bridges. We provide an example of a morphogenetic role for an antibiotic-like molecule.

Amino Acid Sequence↗
PubMed2000

Computational analysis of the first biheterocyclization site of the antibiotic microcin B17.

Microcin B 17 (MccB17) undergoes an enzyme catalyzed posttranslational modification to form four oxazole and four thiazole rings. Four of these rings form 4,2 - connected biheterocyclic functionalities. In this study, the hexapeptide sequence surrounding the first biheterocyclization site of microcin B17 was examined using computational calculations and database analysis to see if it was preorganized for cyclization in a manner similar to that found in the autocatalytic posttranslational cyclization of Green Fluorescent Protein (GFP). Attention was focused on the intermolecular distances between the sulfur and oxygen atoms of the cysteine and serine residues and the carbonyl carbons which they attack in the ring formation. Conformational searches located some low energy conformations that contained relatively short oxygen to carbonyl carbon distances, which indicated that the oxazole forming fragment in microcin B17 is preorganized for cyclization. However, the lack of any clear patterns for the sulfur to carbon distances show that the side-chain of cysteine does not adopt any low energy conformations that are geometrically preorganized for cyclization. The MccB17 synthetase enzyme complex which catalyzes the cyclization process therefore has both steric and electronic functions. The data obtained in this investigation is in agreement with empirical data which shows that biheterocyclization will only occur if the thiazole forms before the oxazole.

Anti-Bacterial Agents↗
PubMed1993

Peptidylglycine alpha-amidating monooxygenase: a multifunctional protein with catalytic, processing, and routing domains.

Peptide alpha-amidation is a widespread, often essential posttranslational modification shared by many bioactive peptides and accomplished by the products of a single gene encoding a multifunctional protein, peptidylglycine alpha-amidating monooxygenase (PAM). PAM has two catalytic domains that work sequentially to produce the final alpha-amidated product peptide. Tissue-specific alternative splicing can generate forms of PAM retaining or lacking a domain required for the posttranslational separation of the two catalytic activities by endoproteases found in neuroendocrine tissue. Tissue-specific alternative splicing also governs the presence of a transmembrane domain and generation of integral membrane or soluble forms of PAM. The COOH-terminal domain of the integral membrane PAM proteins contains routing information essential for the retrieval of PAM from the surface of endocrine and nonendocrine cells. Tissue-specific endoproteolytic processing can generate soluble PAM proteins from integral membrane precursors. Soluble PAM proteins are rapidly secreted from stably transfected nonneuroendocrine cells but are stored in the regulated secretory granules characteristic of neurons and endocrine cells.

Alternative Splicing↗
PubMed1995

Elucidation of the primary structure of the lantibiotic epilancin K7 from Staphylococcus epidermidis K7. Cloning and characterisation of the epilancin-K7-encoding gene and NMR analysis of mature epilancin K7.

Lantibiotics are bacteriocins that contain unusual amino acids such as lanthionines and alpha, beta-didehydro residues generated by posttranslational modification of a ribosomally synthesized precursor protein. The structural gene encoding the novel lantibiotic epilancin K7 from Staphylococcus epidermidis K7 was cloned and its nucleotide sequence was determined. The gene, which was named elkA, codes for a 55-residue preprotein, consisting of an N-terminal 24-residue leader peptide, and a C-terminal 31-residue propeptide which is posttranslationally modified and processed to yield mature epilancin K7. In common with the type-A lantibiotics nisin A and nisin Z, subtilin, epidermin, gallidermin and Pep5, pre-epilancin K7 has a so-called class-Al leader peptide. Downstream and upstream of the elkA gene, the starts of two open-reading-frames, named elkP and elkT, were identified. The elkP and elkT genes presumably encode a leader peptidase and a translocator protein, respectively, which may be involved in the processing and export of epilancin K7. The amino acid sequence of the unmodified pro-epilancin K7, deduced from the elkA gene sequence, is in full agreement with the amino acid sequence of mature epilancin K7, determined previously by means of NMR spectroscopy [van de Kamp, M., Horstink, L. M., van den Hooven, M. W., Konings, R. N. M., Hilbers, C. W., Sahl, H.-G., Metzger, J. W. & van de Ven, F. J. M. (1995) Eur. J. Biochem. 227, 757-771]. The first residue of mature epilancin K7 appears to be modified in a way that has not been described for any other lantibiotic so far. NMR experiments show that the elkA-encoded serine residue at position +1 of pro-epilancin K7 is modified to a 2-hydroxypropionyl residue in the mature protein.

Amino Acid Sequence↗
PubMed1999

Overlapping between fluorescence modifications and activation of prostate transglutaminase induced by sodium dodecyl sulfate.

The transglutaminase from rat coagulating gland secretion has been proposed as a new member of the transglutaminase family. Its basal activity is about 11-fold lower than those of other transglutaminases (e.g., the cytosolic tissue transglutaminase), but reaches levels comparable to those of other transglutaminases on addition of specific surfactant agents. There is no study devoted to understanding the molecular basis of this apparently anomalous activation, which is maximal at approximately 1.5 mM sodium dodecyl sulfate. We provide evidence that in the presence of this detergent modifications of the intrinsic fluorescence as well as energy transfer of the protein fluorescence to a micellar probe parallel the activation of the enzyme. As the sodium dodecyl sulfate concentration inducing maximal activation equals the critical micellar concentration, the biological activity of this transglutaminase appears to be modulated by the binding of micellar aggregates. In fact, the enzyme is modified by posttranslational modifications consisting of some lipid tails. At least two of these tails could act as aggregation nuclei of the enzyme with detergents. This behavior is different from that typical of molecular forms purified from other sources.

Animals↗
PubMed1996

Human p53 expressed in baculovirus-infected Sf9 cells displays a two-dimensional isoform pattern identical to wild-type p53 from human cells.

Baculovirus expression of human p53 protein, a nuclear cell cycle regulator, was examined in Sf9 cells and compared to native p53 synthesized in primary human cells. Maximum expression of the recombinant p53 protein occurred 48 h postinfection. De novo synthesis of the protein was evident for only 2 days postinfection; however, in pulse-chase studies, 30% of the synthesized protein remained stable up to 5 days. Seventy-seven percent of immunoprecipitated, [35S]-methionine-labeled, recombinant p53 protein resided in the cytoplasm of Sf9 cells, while 15% localized to the nucleus and 8% was released extracellularly. Separation of modified p53 protein, by charge and molecular weight, was accomplished by two-dimensional PAGE, and the electrophoretic pattern of the recombinant protein was identical to the wild-type protein from primary human mammary epithelial cells, indicating that the posttranslational modifications of the recombinant protein in this system are similar to those in primary human cells. Eleven isoforms focused between pI 5.75 and pI 6.5. The recombinant p53 isoforms were phosphorylated by 32P-labeling. Phosphatase digestion of immunoprecipitated p53 effectively removed phosphorous groups from the recombinant protein, reducing the number of isoforms from 11 to 2, demonstrating that phosphorylation is the major posttranslational event in the recombinant protein.

Animals↗
PubMed1998

Developmental and hormonal regulation of protein N-glycosylation in the mammary gland.

Glycosylation represents the most common conjugation of both membrane-bound and secreted proteins of animal cells. Among the different types of glycosylation, the N-linked attachment of sugars to the polypeptide backbone is by far the most abundant modification. The biosynthesis of the precursor carbohydrate unit of these proteins is initiated by a stepwise assembly of Glc3Man9GlcNAc2P-P-Dol in the dolichol cycle, its transfer en bloc to the nascent polypeptide in the rough endoplasmic reticulum (RER), followed by excision of the glucosyl residues by processing-specific enzymes, glucosidase I and II, also resident in the endoplasmic reticulum. Additional posttranslational modifications of the carbohydrate moiety in the RER, Golgi, and trans-Golgi network, differ for individual glycoproteins for the completion of final products as high mannose, complex or hybrid glycoproteins en route to their final destinations in the secretory pathway. The enzyme GlcNAc-1-P transferase (GPT) catalyzes the first and committed step, i.e., the transfer of GlcNAc-1-P from UDP-GlcNAc to Dol-P to form GlcNAc-P-P-Dol, in the assembly of the oligosaccharide precursor. Glucosidase I triggers the maturation phase by clipping the distal alpha 1,2-linked Glc residue on the incipient glycoprotein. The critical juxtaposition of the two enzymes in the multistep pathway makes them excellent candidates for the overall regulation of protein N-glycosylation. The highly elevated needs of glycosylation during lactation demand regulation of glycosylation in the gland over and above the levels in the quiescent, virgin and postlactating, regressed gland.

Animals↗
PubMed1998

The role of Gla proteins in vascular calcification.

Arterial calcification occurs with increasing age and in association with a diverse range of diseases, including atherosclerosis, diabetes, and uremia. It occurs at two sites in the vessel wall--in the media where it is known as Monckeberg's sclerosis and in the intima where it is invariably associated with atherosclerosis. Although there are similarities between them, the molecular mechanisms underlying these two forms of calcification may be distinct. Evidence is accumulating that vascular calcification is an active process that has many similarities with ossification, including local expression of bone-associated collagenous and noncollagenous proteins. The recent generation of a matrix gamma-carboxyglutamic acid (Gla) protein (MGP) knockout mouse, which exhibits extensive and lethal calcification and cartilaginous metaplasia of the media of all elastic arteries, has refocused attention on the role of Gla-containing proteins in vascular calcification. Gla-containing proteins have glutamic acid residues that must by gamma-carboxylated by vitamin-K-dependent gamma-carboxylase to enable them to bind calcium and function normally. Therefore, there is considerable scope for both transcriptional and posttranslational modifications of Gla protein function. Recent studies in humans have shown that although MGP mRNA is constitutively expressed by normal vascular smooth muscle cells (VSMCs), it is substantially upregulated in cells adjacent to both medial and intimal calcification. Studies in rats and on cultured human VSMCs showing that inhibition of MGP function by warfarin can accelerate spontaneous calcification have emphasized the potential importance of posttranslational processing in determining MGP function. It is therefore plausible that environmental influences such as diet and medication may have significant effects on vascular calcification. Furthermore, recent studies have shown that several other Gla-containing proteins with the potential to regulate or perhaps contribute to vascular calcification are present in the human vasculature. Future studies on the role of Gla-containing proteins combined with advances in noninvasive imaging techniques to quantify vascular calcification may lead to identification of individuals at particular risk of vascular calcification and the evaluation of novel therapies aimed at regulating its development or progression.

1-Carboxyglutamic Acid↗
PubMed2003

The liberation of CD44.

CD44 was once thought to simply be a transmembrane adhesion molecule that also played a role in the metabolism of its principal ligand hyaluronan. Investigations of CD44 over the past approximately 20 yr have established additional functions for CD44, including its capacity to mediate inflammatory cell function and tumor growth and metastasis. It has also become evident that intricate posttranslational modifications of CD44 regulate the affinity of the receptor for its ligands. In this review, we focus on emerging evidence that functional fragments of the cytoplasmic and ectodomain of CD44 can be liberated by enzymatic modification of cell surfaces as well as of cell-associated matrix. Based on the evidence discussed, we propose that CD44 exists in three phases, as a transmembrane receptor, as an integral component of the matrix, and as a soluble protein found in body fluids, each with biologically significant functions of which some are shared and some distinct. Thus, CD44 represents a model for understanding posttranslational processing and its emerging role as a general mechanism for regulating cell behavior.

Animals↗
PubMed2004

Modification of soybean sucrose synthase by S-thiolation with ENOD40 peptide A.

The gene ENOD40 is expressed at an early stage of root nodule organogenesis and has been postulated to play a central regulatory role in the Rhizobium-legume interaction. In vitro translation of soybean ENOD40 mRNA showed that the gene encodes two peptides of 12 and 24aa residues (peptides A and B) that bind to sucrose synthase. Here we show that the small Cys-containing peptide A binds to sucrose synthase by disulfide bond formation, which may represent a novel form of posttranslational modification of this important metabolic enzyme. Assays using nanomolar concentrations of peptide A revealed that the monomeric reduced form of this peptide binds to purified sucrose synthase. Using a cysteinyl capture strategy combined with MALDI-TOF MS analysis we identified the Cys residue C264 of soybean sucrose synthase as the binding site of peptide A. Modification of sucrose synthase with ENOD40 peptide A activates sucrose cleavage activity whereas the synthesis activity of the enzyme is unaffected. The results are discussed in relation to the role of sucrose synthase in the control of sucrose utilization in nitrogen-fixing nodules.

Amino Acid Substitution↗
PubMed1987

Acetylation of alpha-tubulin in cultured neurons and the induction of alpha-tubulin acetylation in PC12 cells by treatment with nerve growth factor.

alpha-Tubulin of cultured neurons undergoes a posttranslational modification that lowers its p/(Black and Kurdyla, 1983a). We show here that this modification is due to acetylation. Incubation of cultures with 3H-acetate results in the prominent labeling of a protein that comigrates with alpha-tubulin in 1-dimensional SDS gels, and in 2-dimensional isoelectric focusing X SDS gels, it comigrates with the more acidic alpha-tubulin forms. The labeled material can be immunoprecipitated by monoclonal antibodies to alpha-tubulin but not by antibodies to beta-tubulin. The reaction occurs to equal extent in the presence or absence of 0.1 mM cycloheximide or unlabeled amino acids. These observations indicate that the reaction is posttranslational and is not secondary to conversion of isotopic acetate into amino acids. The posttranslational nature of tubulin acetylation was further established by the demonstration that acetate is incorporated into alpha-tubulin in neurites, which lack the capacity for cytoplasmic protein synthesis. Finally, a monoclonal antibody that is specific for acetylated alpha-tubulin (Piperno and Fuller, 1985) recognizes the acidic variants of alpha-tubulin of cultured neurons that can be labeled by incubation with 3H-acetate. Collectively, these observations indicate that alpha-tubulin of cultured sympathetic neurons undergoes a posttranslational acetylation. Immunofluorescence analyses using a monoclonal antibody specific for acetylated alpha-tubulin revealed that it was present throughout the neuron and showed no obvious regional compartmentalization. We have also shown that alpha-tubulin is acetylated in the NGF-responsive PC12 line of pheochromocytoma cells using biochemical and immunological criteria. However, this modification was detected only in long-term (14 d) NGF-treated cells. The results are discussed in terms of the possible role of acetylation as a local mechanism for regulating microtubule stability.

Acetylation↗
PubMed2007

Optimization of cardiac troponin I pull-down by IDM affinity beads and SELDI.

Cardiac troponin I (cTnI) is a key regulator of cardiac muscle contraction. Upon myocardial cell injury, cTnI is lost from the cardiac myocyte and can be detected in serum, in some cases with specific disease-induced modifications, making it an important diagnostic marker for acute myocardial injury. Presently, hospital laboratories use enzyme-linked immunosorbent assays to detect cTnI, but this type of analysis lacks information about modified forms of protein (degradation or phosphorylation) that may give a more specific diagnosis from either serum or biopsies. Because cardiac and serum tissues are widely used for proteomic analysis, it is important to detect these cTnI posttranslational modifications. Therefore, we have chosen to optimize the enrichment and detection of cTnI protein by IDM Affinity Bead pull-down and surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS or SELDI) analyses. By adjusting the chemical compositions of the buffers, we have retained antibody specificity and enriched for different forms of cTnI and its associated proteins.

Animals↗
PubMed1991

Correlation of collagen-linked fluorescence and tendon fiber breaking time.

We review in this paper some reported data on age-dependent modification of proteins pointing out the relationship between the increase of nonenzymatic glycation in abdominal skin collagen of Wistar rats, evaluated by fluorescence intensity, and tendon breaking time, used as a parameter of collagen stiffness. Fluorescence intensity data linearly correlate with the breaking times of collagen fibers from Wistar rats reported from different sources, according to the hypothesis of a common etiological mechanism. It is possible to suppose that posttranslational modifications of proteins play a role in the tissue aging and their level in collagen may be used as a parameter for quantitation.

Aging↗
PubMed2000

Modification of presynaptic CaM kinase II affinity for ATP in hippocampus after long term blockade of serotonin reuptake.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is markedly enriched at synapses, where it is involved in the control of synaptic transmission, transmitter release and synaptic plasticity. CaMKII has also been found to be involved in the long-term action of antidepressants on post-receptor signaling mechanisms, because monoamine reuptake inhibitors induced an increase in autophosphorylation and activity of the kinase in nerve terminals of hippocampus. To study whether changes in the amount of enzyme or kinetic changes, due to posttranslational modifications, are responsible for kinase activation in nerve terminals, alpha-CaMKII level and kinetic constants of the autophosphorylation reaction as a function of ATP concentration were measured in presynaptic cytosol from hippocampus. Treatment with two serotonin reuptake inhibitors did not change the level of presynaptic kinase or the Vmax of autophosphorylation reaction. Instead the Km of the kinase for ATP was decreased 2.8-fold with fluvoxamine and 3.5-fold with paroxetine, implying an increase in the affinity for ATP. This result represents the first finding of changes in kinetic constants of a major brain enzyme after treatment with antidepressant drugs.

Adenosine Triphosphate↗
PubMed2005

Investigating diversity in human plasma proteins.

Plasma proteins represent an important part of the human proteome. Although recent proteomics research efforts focus largely on determining the overall number of proteins circulating in plasma, it is equally important to delineate protein variations among individuals, because they can signal the onset of diseases and be used as biological markers in diagnostics. To date, there has been no systematic proteomics effort to characterize the breadth of structural modifications in individual proteins in the general population. In this work, we have undertaken a population proteomics study to define gene- and protein-level diversity that is encountered in the general population. Twenty-five plasma proteins from a cohort of 96 healthy individuals were investigated through affinity-based mass spectrometric assays. A total of 76 structural forms/variants were observed for the 25 proteins within the samples cohort. Posttranslational modifications were detected in 18 proteins, and point mutations were observed in 4 proteins. The frequency of occurrence of these variations was wide-ranged, with some modifications being observed in only one sample, and others detected in all 96 samples. Even though a relatively small cohort of individuals was investigated, the results from this study illustrate the extent of protein diversity in the human population and can be of immediate aid in clinical proteomics/biomarker studies by laying a basal-level statistical foundation from which protein diversity relating to disease can be evaluated.

Adolescent↗
PubMed2002

High glucose and insulin promote O-GlcNAc modification of proteins, including alpha-tubulin.

Increased flux through the hexosamine biosynthesis pathway has been implicated in the development of glucose-induced insulin resistance and may promote the modification of certain proteins with O-linked N-acetylglucosamine (O-GlcNAc). L6 myotubes (a model of skeletal muscle) were incubated for 18 h in 5 or 25 mM glucose with or without 10 nM insulin. As assessed by immunoblotting with an O-GlcNAc-specific antibody, high glucose and/or insulin enhanced O-GlcNAcylation of numerous proteins, including the transcription factor Sp1, a known substrate for this modification. To identify novel proteins that may be O-GlcNAc modified in a glucose concentration/insulin-responsive manner, total cell membranes were separated by one- or two-dimensional gel electrophoresis. Selected O-GlcNAcylated proteins were identified by mass spectrometry (MS) analysis. MS sequencing of tryptic peptides identified member(s) of the heat shock protein 70 (HSP70) family and rat alpha-tubulin. Immunoprecipitation/immunoblot studies demonstrated several HSP70 isoforms and/or posttranslational modifications, some with selectively enhanced O-GlcNAcylation following exposure to high glucose plus insulin. In conclusion, in L6 myotubes, Sp1, membrane-associated HSP70, and alpha-tubulin are O-GlcNAcylated; the modification is markedly enhanced by sustained increased glucose flux.

Acetylglucosamine↗