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PubMed1995

A novel strategy to identify maternal and paternal inheritance in the mouse.

A novel strategy for identifying proteins which reveal maternal or paternal inheritance in the mouse is presented. Using two-dimensional electrophoresis we investigated protein expression patterns of adult liver and different embryonic and extraembryonic tissue in C57BL/6Crl and in DBA/2Crl mice, as well as in their reciprocal hybrids. We found three groups of protein spots which showed maternal or paternal inheritance of quantitative variations. These proteins were characterized by N-terminal or internal amino acid sequencing, by determination of the amino acid composition, by glycoprotein staining and RNA expression analysis. The three proteins identified were: alpha-enolase, cyclophilin and beta-group hemoglobins. The parental effects observed for alpha-enolase and cyclophilin were found to be due to parent-specific post-translational modifications of these proteins. For the beta-group hemoglobins our results suggested parental effects on the transcriptional level.

Amino Acid Isomerases↗
PubMed1990

Fatty acids on the A/Japan/305/57 influenza virus hemagglutinin have a role in membrane fusion.

The covalent attachment of fatty acid moieties to proteins is a widespread post-translational modification of viral and cell proteins yet the functional consequences of acylation are not well understood. We have determined that the A/Japan/305/57 influenza virus hemagglutinin (HA) contains three potential acylation sites at cysteine residues 211, 218 and 221 in the cytoplasmic domain of the molecule. Site-directed mutagenesis of one or more of these sites has no effect on biosynthesis, transport or receptor binding activity of the molecule; however, modification of any single site is sufficient to abolish completely or inhibit severely membrane fusion activity, a function essential for virus infectivity. We present a molecular model of the transmembrane and cytoplasmic domains of the HA to illustrate the potential orientation of these fatty acids and to provide a conceptual framework for further experimentation.

Acylation↗
PubMed2000

N-glycosylation and residue 96 are involved in the functional properties of UDP-glucuronosyltransferase enzymes.

The recent cloning of several human and monkey UDP-glucuronosyltransferase (UGT) 2B proteins has allowed the characterization of these steroid metabolic enzymes. However, relatively little is known about the structure-function relationship, and the potential post-translational modifications of these proteins. The mammalian UGT2B proteins contain at least one consensus asparagine-linked glycosylation site NX(S/T). Endoglycosidase H digestion of the human and monkey UGT2B proteins demonstrates that only UGT2B7, UGT2B15, UGT2B17, and UGT2B20 are glycosylated. Although UGT2B15 and UGT2B20 contain three and four potential glycosylation sites, respectively, site-directed mutagenesis revealed that both proteins are glycosylated at the same first site. In both proteins, abolishing glycosylation decreased glucuronidation activity; however, the K(m) values and the substrate specificities were not affected. Despite the similarities between UGT2B15 and UGT2B20, UGT2B20 is largely more labile than UGT2B15. Treating HK293 cells stably expressing UGT2B20 with cycloheximide for 2 h decreased the enzyme activity by more than 50%, whereas the activity of UGT2B15 remained unchanged after 24 h. The UGT2B20 protein is unique in having an isoleucine at position 96 instead of an arginine as found in all the other UGT2B enzymes. Changing the isoleucine in UGT2B20 to an arginine stabilized enzyme activity, while the reciprocal mutation in UGT2B15 R96I produced a more labile enzyme. Secondary structure predictions of UGT2B proteins revealed a putative alpha-helix in this region in all the human and monkey proteins. This alpha-helix is shortest in UGT2B20; however, the helix is lengthened in UGT2B20 I96R. Thus, it is apparent that the length of the putative alpha-helix between residues 84 and 100 is a determining factor in the stability of UGT2B enzyme activity. This study reveals the extent and importance of protein glycosylation on UGT2B enzyme activity and that the effect of residue 96 on UGT2B enzyme stability is correlated to the length of a putative alpha-helix.

Amino Acid Sequence↗
PubMed1989

Expression of the kinase domain of mouse protein kinase C in E. coli.

The kinase domain of mouse protein kinase C type alpha has been expressed at high levels in E. coli. The protein has been purified 500-fold taking advantage of the fact that highly expressed fusion proteins precipitate out in the bacterial cell and can be solubilized in 7 M urea. The purified protein can be detected with an antibody generated against a PKC alpha derived synthetic peptide. The purified kinase domain exhibits no measurable kinase activity in a protamin phosphorylation assay. This could be an indication that post-translational modifications of the protein kinase domain which do not happen in bacteria are a requirement for functional enzyme activity or that the regulatory domain of protein kinase C is indispensable for kinase function.

Animals↗
PubMed1995

Copper binding to the N-terminal tandem repeat regions of mammalian and avian prion protein.

Mammalian prion protein (PrP) is a normal cellular protein (PrPc) which through post-translational modification produces the infectious prion protein (PrPsc). We have shown, using mass spectrometry, that synthetic peptides containing three or four copies of an octapeptide repeat sequence (PHGGGWGQ), found in a highly conserved N-terminal domain of PrP, preferentially bind copper over other metals. Peptides from the analogous region of chicken PrP, which contains an N-terminal repeat domain of the hexapeptide (NPGYPH), showed similar specificity for copper binding. In addition, gel filtration chromatography demonstrated concentration dependent binding of copper to the mammalian tetra repeat PrP peptide. These results suggest that PrP may be a copper binding protein in vivo.

Amino Acid Sequence↗
PubMed2006

Derivatized nanoparticle coated capillaries for purification and micro-extraction of proteins and peptides.

Various methods are used to enrich or purify a protein of interest from other proteins and components in a crude cell lysate or other sample. One of the most powerful methods is affinity purification, also called affinity chromatography, whereby the proteins of interest are purified by virtue of their specific binding properties to an immobilized ligand. Affinity purification is becoming more widely used for exploring post-translation modifications and protein-protein interactions, especially with a view toward developing new general tag systems and strategies of chemical derivatization on peptides for affinity selection. Our work was aimed to immobilize proteins or ligands for affinity purification of antibodies, fusion-tagged proteins and other proteins and peptides. Selected proteins or peptides are efficiently extracted and enriched using chemically derivatized walls of a fused silica capillary column. In this paper, we present an open tubular capillary, where the inner wall of a fused silica capillary was derivatized by covalent binding of modified polystyrene latex particles. The capillaries were derivatized with iminodiacetic acid and loaded with Fe3+ or Ni2+ for the purification and enrichment of phosphopeptides or His-tagged proteins, respectively. The latex coated capillaries have been successfully applied to enrich phosphopeptides from beta-casein tryptic digest and ovalbumin tryptic digest at a micro volume scale with recoveries ranging from 92 to 95%. The capillaries have been eluted under conditions compatible with MALDI-MS without any prior desalting step. In another approach, concanavalin A (Con A) or Protein G were immobilized on the epoxy modified latex on the inner wall of the fused silica capillary for the purification of glycoproteins and immunoglobulin, respectively. The design of the capillary and the protocols used for purification permits the direct detection of eluted proteins and peptides with gel electrophoresis or with mass spectrometry. The elution volumes are passed as discrete segments of few microliters over the inner surface of the open-tube capillary, achieving enrichment factors of more than 20-fold from starting samples.

Caseins↗
PubMed2005

Effects of over-expression of the regulatory enzymes DraT and DraG on the ammonium-dependent post-translational regulation of nitrogenase reductase in Azospirillum brasilense.

Nitrogen fixation in Azospirillum brasilense is regulated at transcriptional and post-translational levels. Post-translational control occurs through the reversible ADP-ribosylation of dinitrogenase reductase (Fe Protein), mediated by the dinitrogenase reductase ADP-ribosyltransferase (DraT) and dinitrogenase reductase glycohydrolase (DraG). Although the DraT and DraG activities are regulated in vivo, the molecules responsible for such regulation remain unknown. We have constructed broad-host-range plasmids capable of over-expressing, upon IPTG induction, the regulatory enzymes DraT and DraG as six-histidine-N-terminal fused proteins (His). Both DraT-His and DraG-His are functional in vivo. We have analyzed the effects of DraT-His and DraG-His over-expression on the post-translational modification of Fe Protein. The DraT-His over-expression led to Fe Protein modification in the absence of ammonium addition, while cells over-expressing DraG-His showed only partial ADP-ribosylation of Fe Protein by adding ammonium. These results suggest that both DraT-His and DraG-His lose their regulation upon over-expression, possible by titrating out negative regulators.

ADP Ribose Transferases↗
PubMed1998

Electrophoresis combined with novel mass spectrometry techniques: powerful tools for the analysis of proteins and proteomes.

Analytical and preparative electrophoresis separation techniques have been essential tools in protein biochemistry and the biological sciences in general. The combination of high resolution electrophoresis techniques with high performance analytical procedures has dramatically enhanced analytical protein biochemistry. In this report we describe the combination of electrophoretic separation techniques with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). A series of different techniques, consisting of automated high performance liquid chromatography (HPLC)-MS/MS, capillary-HPLC-MS/MS, and solid phase extraction (SPE)-capillary zone electrophoresis (CZE)-MS/MS, are described in the context of the identification of high pmol to the low fmol amounts of proteins. Application of these powerful new tools for the analysis of proteins on a large proteome-wide scale is presented. Furthermore, the combination of orthogonal separation techniques, such as immobilized metal affinity chromatography (IMAC) with SPE-CZE-MS/MS, and IMAC followed by HPLC, and by SPE-CZE-MS/MS, are presented for the detailed investigation of post-translational modifications of specific proteins.

Amino Acid Sequence↗
PubMed2002

Telomerase regulation: not just flipping the switch.

Significant research efforts have focused on the transcriptional regulation of the catalytic protein component of telomerase, hTERT (human telomerase reverse transcriptase). However, increasing evidence indicates that regulation of hTERT is complex and not as simple as just flipping a switch. Although much remains to be learned about the regulation of telomerase, generation of full-length hTERT protein is likely to be the limiting step in the formation of active telomerase. Regulation of hTERT transcription, transport to the nucleus, assembly of the telomerase holoenzyme, recruitment of telomerase to the telomere, and the role of post-translational modifications of hTERT protein are areas of intense investigation. It is hoped that shedding light on these diverse areas of telomerase regulation will allow us to manipulate telomerase activity for therapeutic purposes.

Active Transport, Cell Nucleus↗
PubMed1979

Proteins specified by togaviruses in infected Aedes albopictus (Singh) mosquito cells.

Yields of greater than 10(7) p.f.u./ml at 28 or 37 degrees C of the alphavirus Sindbis and the flavivirus Kunjin were obtained in the Aedes Albopictus (Singh) cell line, the latent periods being 4 to 6 and 10 to 12 h, respectively. Despite a high background of host protein synthesis, virtually all the virus-specified proteins of the flaviviruses Kunjin, Dengue-2 and Japanese encephalitis were labelled and resolved by slab gel electrophoresis of infected and uninfected cell proteins. In contrast, only one induced protein, of mol. wt. 30 000, was identified in cells labelled during Sindbis virus infection. The envelope glycoprotein V3 of Kunjin virus was resolved as a double band in samples of infected cytoplasm labelled with 3H-glucosamine, similar to that of carbohydrate-labelled V3 in vertebrate (Vero) cells. Attempts to reduce host protein synthesis selectively during labelling periods were unsuccessful using either a hypertonic inhibition block or treatment with 0.1 mu g actinomycin D per ml. The most efficient labelling of Kunjin virus-specified proteins was achieved at 37 degrees C in the presence of actinomycin D. The largest non-structural flavivirus protein NV5 migrated slightly faster than NV5 from infected vertebrate (Vero) cells. The small non-structural proteins NV1, NV1 1/2 and NV2 from infected mosquito cells were successively trimmed during post translational periods exceeding 70 min, compared to much shorter periods reported previously for post translational modifications of these proteins in vertebrate cells.

Aedes↗
PubMed2005

ROXY1, a member of the plant glutaredoxin family, is required for petal development in Arabidopsis thaliana.

We isolated three alleles of an Arabidopsis thaliana gene named ROXY1, which initiates a reduced number of petal primordia and exhibits abnormalities during further petal development. The defects are restricted to the second whorl of the flower and independent of organ identity. ROXY1 belongs to a subgroup of glutaredoxins that are specific for higher plants and we present data on the first characterization of a mutant from this large Arabidopsis gene family for which information is scarce. ROXY1 is predominantly expressed in tissues that give rise to new flower primordia, including petal precursor cells and petal primordia. Occasionally, filamentous organs with stigmatic structures are formed in the second whorl of the roxy1 mutant, indicative for an ectopic function of the class C gene AGAMOUS (AG). The function of ROXY1 in the negative regulation of AG is corroborated by premature and ectopic AG expression in roxy1-3 ap1-10 double mutants, as well as by enhanced first whorl carpeloidy in double mutants of roxy1 with repressors of AG, such as ap2 or lug. Glutaredoxins are oxidoreductases that oxidize or reduce conserved cysteine-containing motifs. Mutagenesis of conserved cysteines within the ROXY1 protein demonstrates the importance of cysteine 49 for its function. Our data demonstrate that, unexpectedly, a plant glutaredoxin is involved in flower development, probably by mediating post-translational modifications of target proteins required for normal petal organ initiation and morphogenesis.

AGAMOUS Protein, Arabidopsis↗
PubMed1988

Extensive labeling with [3H]ethanolamine of a hydrophilic protein of animal cells.

Murine T-lymphomas and Thy-1- mutants were labeled overnight with [3H]ethanolamine to detect proteins which possess a glycophospholipid anchor. When labeled cells were treated with 10% trichloroacetic acid and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, both Thy-1 and a second intensely labeled protein (46 kDa) were observed. The presence of the radiolabeled 46-kDa protein in wild type and class E Thy-1 negative cells (cells in which Thy-1 is synthesized but cannot be labeled with [3H]ethanolamine) suggested incorporation into a distinct moiety. Labeling of the 46-kDa protein with [3H]ethanolamine is rapidly inhibited by cycloheximide. Further characterization of the 46-kDa protein by subcellular fractionation and Triton X-114 partitioning indicated that the protein is located in the cytosol. The protein is basic and does not bind to either concanavalin A or wheat germ agglutinin. Labeling of a 46-kDa protein has also been demonstrated in Chinese hamster ovary, COS, rat myeloma, cloned human T-lymphocytes, and HeLa cells. Pronase digestion of the [3H]ethanolamine-labeled 46-kDa protein of wild type lymphoma cells generated a nonbasic and polar labeled fragment which is labile to strong acid and base ([3H]ethanolamine is liberated), insensitive to periodate oxidation and alkaline phosphatase, and does not bind to concanavalin A or wheat germ agglutinin. Judging from methylation studies, the labeled ethanolamine residue does not contain a free amino group. Based on these results, we report a novel post-translational modification of selected protein(s) by the covalent addition of [3H]ethanolamine.

Amino Acids↗
PubMed1997

Recombinant human extracellular superoxide dismutase produced in milk of transgenic rabbits.

Expression of human extracellular superoxide dismutase (EC-SOD), a glycosylated, tetrameric metalloprotein, was targeted to the lactating mammary gland of transgenic rabbits. Efficient expression of the recombinant whey acidic protein/ec-sod gene was achieved and up to 3 mg ml-1 of the enzyme was secreted into the milk. Rabbit milk-produced recombinant EC-SOD was primarily found in the whey and purified by a two-step chromatographic method. To evaluate the rabbit milk-produced human EC-SOD, comparisons with native and Chinese hamster ovary cell (CHO)-produced EC-SOD were performed. All proteins were tetrameric and N-glycosylated. The behaviour on SDS-PAGE and size-exclusion chromatography indicated that the masses, and thereby the extent of post-translational modification of the proteins was similar. The monosaccharide composition of both recombinant EC-SOD variants was analysed and indicated similarities in the attached N-glycans on the two proteins. Furthermore, the peptide maps of the three EC-SOD variants revealed that all proteins had similar polypeptide backbones.

Animals↗
PubMed1994

Association of casein kinase 2 with nuclear matrix. Possible role in nuclear matrix protein phosphorylation.

The nuclear matrix is thought to play a fundamental role in the nuclear structure and functions relating to cell proliferation and regulation of gene activity. Phosphorylation of nuclear matrix proteins has been observed, and multiple protein kinases may be involved in this process. Casein kinase 2 is a ubiquitous messenger-independent serine/threonine protein kinase that has been implicated in cellular growth and proliferation. The presence of immunoreactive casein kinase 2 in purified nuclear matrix preparations was demonstrated in rat liver and prostate tissues by employing specific antibodies. The enzyme was active toward the specific decapeptide substrate Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu and exhibited other properties characteristic of casein kinase 2. Phosphorylation of nuclear matrix proteins catalyzed by intrinsic protein kinase activity in intact nuclei was also observed, and the results suggested that this reaction was at least in part catalyzed by casein kinase 2. The profile of nuclear matrix proteins labeled with 32Pi in situ in intact liver cells was similar to that observed in nuclear matrix proteins isolated from nuclei labeled with [gamma-32P]ATP in vitro. Association of casein kinase 2 with the nuclear matrix may be of significance in the post-translational modification of certain proteins in this fraction.

Amino Acid Sequence↗
PubMed2004

Automated, on-line two-dimensional nano liquid chromatography tandem mass spectrometry for rapid analysis of complex protein digests.

Evaluation of cellular processes and their changes at the level of protein expression and post-translational modifications may allow identification of novel proteins and the mechanisms involved in pathogenic processes. However, the number of proteins and, after tryptic digestion, of peptides from a single cell can be tremendously high. Separation and analysis of such complex peptide mixtures can be performed using multidimensional separation techniques such as two-dimensional gel electrophoresis or two-dimensional-high-performance liquid chromatography (2-D-HPLC). The aim of this work was to establish a fully automated on-line 2-D-HPLC separation method with column switching for the separation of complex tryptic digests. A model mixture of five proteins as well as a nuclear matrix protein sample were digested with trypsin and separated using a strong cation exchange (SCX) column in the first dimension and nano reversed phase in the second dimension. Separated peptides were detected using an ion trap mass spectrometer. The advantages of this new fully automated method are rapid sample loading, the possibility of injecting large volumes and no introduction of salt into the mass spectrometer. Furthermore, column switching allows the independent control and optimization of the two dimensions independently.

Animals↗
PubMed2006

Use of analogs and inhibitors to study the functional significance of protein palmitoylation.

Covalent attachment of palmitate to proteins is a post-translational modification that exerts diverse effects on protein localization and function. The three key technical approaches required for an investigator to determine the role of palmitoylation of your favorite palmitoylated protein (YFPP) are methods to: (1) detect YFPP palmitoylation; (2) alter or inhibit palmitoylation of YFPP; (3) determine the functional significance of altered YFPP palmitoylation. Here, I describe experimental methods to address these three issues. Both radioactive (radiolabeling with [(3)H]palmitate or (125)I-IC16 palmitate) and non-radioactive (chemical labeling and mass spectrometry) methods to detect palmitoylated proteins are presented. Next, techniques to inhibit protein palmitoylation are described. These include site specific mutagenesis, and treatment of cells with inhibitors of protein palmitoylation, including 2-bromopalmitate, cerulenin, and tunicamycin. Alternative methods to replace palmitate with other fatty acids are also presented. Finally, general approaches to determining the effect of altered palmitoylation status on YFPP association with membranes and lipid rafts, as well as signal transduction, are described.

Acylation↗
PubMed2002

Intracellular glycosylation and development.

O-linked N-acetylglucosamine (O-GlcNAc) is a highly dynamic post-translational modification of cytoplasmic and nuclear proteins. Although the function of this abundant modification is yet to be definitively elucidated, all O-GlcNAc proteins are phosphoproteins. Further, the serine and threonine residues substituted with O-GlcNAc are often sites of, or close to sites of, protein phosphorylation. This implies that there may be a dynamic interplay between these two post-translational modifications to regulate protein function. In this review, the functions of some of the proteins that are modified by O-GlcNAc will be considered in the context of the potential role of the O-GlcNAc modification. Furthermore, predictions will be made as to how cellular function and developmental regulation might be affected by changes in O-GlcNAc levels.

Animals↗