The B cell-specific major raft protein, Raftlin, is necessary for the integrity of lipid raft and BCR signal transduction.
Recent evidence indicates that membrane microdomains, termed lipid rafts, have a role in B-cell activation as platforms for B-cell antigen receptor (BCR) signal initiation. To gain an insight into the possible functioning of lipid rafts in B cells, we applied liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) methodologies to the identification of proteins that co-purified with lipid rafts of Raji cells. Among these raft proteins, we characterized a novel protein termed Raftlin (raft-linking protein). Like the Src family kinase, Raftlin is localized exclusively in lipid rafts by fatty acylation of N-terminal Gly2 and Cys3, and is co-localized with BCR before and after BCR stimulation. Disruption of the Raftlin gene in the DT40 B-cell line resulted in a marked reduction in the quantity of lipid raft components, including Lyn and ganglioside GM1, while overexpression of Raftlin increased the content of raft protein. Moreover, BCR-mediated tyrosine phosphorylation and calcium mobilization were impaired by the lack of Raftlin and actually potentiated by overexpression of Raftlin. These data suggest that Raftlin plays a pivotal role in the formation and/or maintenance of lipid rafts, therefore regulating BCR-mediated signaling.


Processing of the gastrin precursor. Modulation of phosphorylated, sulfated, and amidated products.
Post-translational processing of the precursor for rat gastrin yields products that include peptides phosphorylated at Ser96, amidated at Phe92, and sulfated at Tyr87 or Tyr103. The phosphorylation site is immediately adjacent to the processing point that gives rise to the biologically active amidated gastrins. We have examined changes in post-translational processing which occur in gastrin cells from rats that are physiologically stimulated (by feeding) or unstimulated (by fasting). Peptides were identified using site-directed radioimmunoassays and chromatographic systems that resolve phosphorylated, amidated, and sulfated progastrin products, including intermediates generated prior to amidation (i.e. C-terminal glycine-extended variants). Assays for Phe92-amidated peptides and for the C-terminal tryptic fragment of progastrin indicated decreases in the total tissue concentrations of immunoreactive peptide with fasting; in contrast, the tissue concentrations of glycine-extended biosynthetic intermediates were similar in fasted and fed rats. Taken together the data suggest a relative failure in amidation mechanisms in unstimulated cells. The endopeptidase cleavage of progastrin was not influenced significantly by fasting. However, the phosphorylation of peptide products containing Ser96 was depressed significantly in fasted rats. The proportions of amidated peptides sulfated at Tyr87 were generally lower than their corresponding glycine-extended biosynthetic precursors, but in both cases the proportion of peptide in the sulfated form was lower than for peptides sulfated at Tyr103. Feeding did not change the sulfation of amidated heptadecapeptide gastrin or its glycine-extended variant. The results suggest that the mechanisms determining phosphorylation and amidation of progastrin-related peptides depend on the patterns of stimulation of gastrin cells. The observation that decreased phosphorylation is associated with a failure to produce active amidated products is consistent with a regulatory function for phosphorylation in gastrin production.


NEDD8 acts as a 'molecular switch' defining the functional selectivity of VHL.
The von Hippel-Lindau (VHL) tumour suppressor protein is important in the E3 ubiquitin ligase ECV (Elongin B/C-CUL2-VHL)-mediated destruction of hypoxia-inducible factor and the promotion of fibronectin (FN) extracellular matrix assembly. Although the precise molecular mechanism controlling the selectivity of VHL function remains unknown, a failure in either process is associated with oncogenic progression. Here, we show that VHL performs its FN-associated function independently of the ECV complex, highlighting the autonomy of these pathways. Furthermore, we show that NEDD8, a ubiquitin-like molecule, acts as a 'molecular switch' in which its covalent conjugation to VHL prohibits the engagement of the scaffold component CUL2 and, concomitantly, activates the association with FN. These findings provide the first mechanistic step in defining the functional selectivity of VHL and explain a previously unrecognized function of NEDD8.


Understanding GFP posttranslational chemistry: structures of designed variants that achieve backbone fragmentation, hydrolysis, and decarboxylation.
The green fluorescent protein (GFP) creates a fluorophore out of three sequential amino acids by promoting spontaneous posttranslational modifications. Here, we use high-resolution crystallography to characterize GFP variants that not only undergo peptide backbone cyclization but additional denaturation-induced peptide backbone fragmentation, native peptide hydrolysis, and decarboxylation reactions. Our analyses indicate that architectural features that favor GFP peptide cyclization also drive peptide hydrolysis. These results are relevant for the maturation pathways of GFP homologues, such as the kindling fluorescent protein and the Kaede protein, which use backbone cleavage to red-shift the spectral properties of their chromophores. We further propose a photochemical mechanism for the decarboxylation reaction, supporting a role for the GFP protein environment in facilitating radical formation and one-electron chemistry, which may be important in activating oxygen for the oxidation step of chromophore biosynthesis. Together, our results characterize GFP posttranslational modification chemistry with implications for the energetic landscape of backbone cyclization and subsequent reactions, and for the rational design of predetermined spontaneous backbone cyclization and cleavage reactions.


Molecular cloning and characterization of nlpH, encoding a novel, surface-exposed, polymorphic, plasmid-encoded 33-kilodalton lipoprotein of Borrelia afzelii.
Borrelia burgdorferi sensu lato organisms, comprising B. burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii, are tick-borne pathogens causing Lyme borreliosis in humans. To identify putative virulence determinants, a B. afzelii DNA library was screened for Congo red dye binding, a property associated with virulence in pathogenic bacteria. One clone was found to carry a 663-nucleotide-long open reading frame encoding a Congo red dye-binding protein with a calculated molecular mass of 25,660 Da. The amino acid sequence deduced from its nucleotide sequence was found to include a consensus bacterial lipidation site present at residues 15 to 18 (Leu-Ser-Gly-Cys). The lipoprotein nature was demonstrated by incorporation of radioactive palmitate; hence, this protein has been termed NlpH, for new lipoprotein H. NlpH is located on the surface of B. afzelii, and the nlpH gene is found on a circular plasmid. The nlpH gene is also found in B. burgdorferi sensu stricto and B. garinii. Immediately upstream of nlpH is located a smaller reading frame encoding a polypeptide containing the casein kinase II phosphorylation recognition sequence, (Ser/Thr)-X-Y-(Glu/Asp), repeated 10 times.


Effect of metal binding and posttranslational lysine carboxylation on the activity of recombinant hydantoinase.
Bacterial hydantoinase possesses a binuclear metal center in which two metal ions are bridged by a posttranslationally carboxylated lysine. How the carboxylated lysine and metal binding affect the activity of hydantoinase was investigated. A significant amount of iron was always found in Agrobacterium radiobacter hydantoinase purified from unsupplemented cobalt-, manganese-, or zinc-amended Escherichia coli cell cultures. A titration curve for the reactivation of apohydantoinase with cobalt indicates that the first metal was preferentially bound but did not give any enzyme activity until the second metal was also attached to the hydantoinase. The pH profiles of the metal-reconstituted hydantoinase were dependent on the specific metal ion bound to the active site, indicating a direct involvement of metal in catalysis. Mutation of the metal binding site residues, H57A, H59A, K148A, H181A, H237A, and D313A, completely abolished hydantoinase activity but preserved about half of the metal content, except for K148A, which lost both metals in its active site. However, the activity of K148A could be chemically rescued by short-chain carboxylic acids in the presence of cobalt, indicating that the carboxylated lysine was needed to coordinate the binuclear ion within the active site of hydantoinase. The mutant D313E enzyme was also active but resulted in a pH profile different from that of wild-type hydantoinase. A mechanism for hydantoinase involving metal, carboxylated K148, and D313 was proposed.


The cataract-associated R14C mutant of human gamma D-crystallin shows a variety of intermolecular disulfide cross-links: a Raman spectroscopic study.
The Arg14 to Cys (R14C) mutation in the human gammaD-crystallin (HGD) gene has been associated with a juvenile-onset hereditary cataract. We showed previously [Pande, A., et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 1993-1998] that rapid oxidation of Cys14 in the mutant leads to the formation of intermolecular, disulfide-cross-linked aggregates at physiological pH. Here we present a Raman spectroscopic analysis of R14C and HGD and show that R14C forms such aggregates even at pH 4.5. The lower pH enabled us to monitor the evolution of a variety of disulfide cross-links with distinct conformations around the CC-SS-CC dihedral angles. At least three cysteine residues are involved, forming protein-protein cross-links through disulfide-exchange reactions. From the pattern of the S-S and Trp Raman bands, we infer that Cys32 is likely to be involved in the cross-linking. The data suggest that protein precipitation in the mutant may not be the direct result of disulfide cross-linking, although such cross-linking is the initiating event. Thus, our Raman data not only enhance the understanding of the reactivity of Cys14 in the R14C mutant and the mechanism of opacity, but also shed light on the mechanism of oxidative degradation during long-term storage of thiol-containing pharmaceuticals.


SUMO modification enhances p66-mediated transcriptional repression of the Mi-2/NuRD complex.
Human p66alpha and p66beta are two potent transcriptional repressors that interact with the methyl-CpG-binding domain proteins MBD2 and MBD3. An analysis of the molecular mechanisms mediating repression resulted in the identification of two major repression domains in p66alpha and one in p66beta. Both p66alpha and p66beta are SUMO-modified in vivo: p66alpha at two sites (Lys-30 and Lys-487) and p66beta at one site (Lys-33). Expression of SUMO1 enhanced the transcriptional repression activity of Gal-p66alpha and Gal-p66beta. Mutation of the SUMO modification sites or using a SUMO1 mutant or a dominant negative Ubc9 ligase resulted in a significant decrease of the transcriptional repression of p66alpha and p66beta. The Mi-2/NuRD components MBD3, RbAp46, RbAp48, and HDAC1 were found to bind to both p66alpha and p66beta in vivo. Most of the interactions were not affected by the SUMO site mutations in p66alpha or p66beta, with two exceptions. HDAC1 binding to p66alpha was lost in the case of a p66alphaK30R mutant, and RbAp46 binding was reduced in the case of a p66betaK33R mutant. These results suggest that interactions within the Mi-2/NuRD complex as well as optimal repression are mediated by SUMOylation.


Prenylated isoforms of yeast casein kinase I, including the novel Yck3p, suppress the gcs1 blockage of cell proliferation from stationary phase.
The GCS1 gene of the budding yeast Saccharomyces cerevisiae mediate the resumption of cell proliferation from the starved, stationary-phase state. Here we identify yeast genes that, in increased dosages, overcome the growth defect of gcs1 delta mutant cells. Among these are YCK1 (CK12) and YCK2 (CKI1), encoding membrane-associated casein kinase I, and YCK3, encoding a novel casein kinase I isoform. Some Yck3p gene product was found associated with the plasma membrane, like Yck1p and Yck2p, but most confractionated with the nucleus, like another yeast casein kinase I isoform, Hrr25p. Genetic studies showed that YCK3 and HRR25 constitute an essential gene family and that Yck3p can weakly substitute for Yck1p-Yck2p. For gcs1 delta suppression, both a protein kinase domain and a C-terminal prenylation motif were shown to be necessary. An impairment in endocytosis was found for gcs1 delta mutant cells, which was alleviated by an increased YCK2 gene dosage. The ability of an increased casein kinase I gene dosage to suppress the effects caused by the absence of Gcs1p suggests that Gcs1p and Yck1p-Yck2p affect parallel pathways.


Spectroscopic analysis of acylated and deacylated myelin proteolipid protein.
The effect of covalently bound fatty acid on the conformation of the myelin proteolipid protein has been studied by ultraviolet and intrinsic fluorescence spectroscopy. With dimethyl sulfoxide used as a perturbant, the exposure of Trp and Tyr residues in various mixtures of chloroform-methanol was evaluated by difference spectroscopy of the proteolipid protein (APL) and its chemically deacylated form (d-APL). The fraction of chromophoric groups exposed increased with the proportion of chloroform with 25% of the groups exposed in 1:2 chloroform-methanol and 98% in 3:1 chloroform-methanol. These conformational changes correlate well with changes in intrinsic viscosity. Values for the deacylated form were indistinguishable from those of the acylated protein, suggesting that fatty acids do not affect protein conformation in organic solvents. In water, UV difference spectroscopy indicated that the number of Tyr and Trp groups exposed in both APL and d-APL was relatively small and was independent of the molecular size of the perturbant. However, differences in the environment of the Trp groups in the two forms of the protein could be demonstrated by intrinsic fluorescence. When the protein was excited at 295 nm, the maximum emission wavelength for the acylated protein was 330 nm, whereas it was 335 nm for the deacylated form. Furthermore, the Trp groups in d-APL were more easily quenched by acrylamide than in APL, indicating that they were more exposed, or in a more hydrophilic environment, following deacylation. Protein aggregation appears to be independent of the presence of fatty acids, suggesting that the fluorescence differences between APL and d-APL are related to factors other than aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)


The 31-kDa precursor of interleukin 1 alpha is myristoylated on specific lysines within the 16-kDa N-terminal propiece.
The cytokine interleukin 1 alpha (IL-1 alpha) is a critical mediator of the immune and inflammatory responses. A unique determinant of its activity as compared with IL-1 beta may be its association with the plasma membrane. While the biologic activity of "membrane IL-1" has been extensively reported, the mechanism of membrane binding remains unclear. We report that the N terminus of the 31-kDa IL-1 alpha precursor is myristoylated on specific internal lysine residues. Immunoprecipitation of [3H]myristic acid-radiolabeled human monocyte lysates with IgG antibodies to the 31-kDa IL-1 alpha precursor recovered a protein with the physicochemical properties of the IL-1 alpha N-terminal propiece (16 kDa, pI 4.45). Glycyl N-myristoylation of this protein is precluded by the absence of a glycine residue at position 2, suggesting that the propiece is myristoylated on epsilon-amino groups of lysine. To determine which lysine(s) are acylated, a series of synthetic peptides containing all lysines found in the IL-1 alpha N-terminal propiece were used in an in vitro myristoylation assay containing peptide, myristoyl-CoA, and monocyte lysate as enzyme source. Analysis of the reaction products by reverse-phase HPLC and gas-phase sequencing demonstrated the specific myristoylation of Lys-82 and Lys-83, yielding predominantly monoacylated product. A conserved sequence in the IL-1 beta propiece was myristoylated with at least 8-fold less efficiency. Acylation of the IL-1 alpha precursor by a previously unrecognized lysyl epsilon-amino N-myristoyl-transferase activity may facilitate its specific membrane targeting.


HIV-1 Nef alters the expression of betaII and epsilon isoforms of protein kinase C and the activation of the long terminal repeat promoter in human astrocytoma cells.
In the human immunodeficiency virus type 1 (HIV-1)-infected brain, the virus does not replicate in astrocytes, but a synthesis of viral regulatory proteins occurs in these cells, leading to accumulation of Nef. As an approach to understand the effects of Nef on astrocyte functional activity, we analyzed whether intracellular Nef interferes with the expression and activation of the enzyme protein kinase C (PKC), which is an important regulator of astroglial functions and HIV-1 replication. Astrocytoma clones (U251 MG) not expressing Nef (Neo), or expressing wild-type Nef (Bru) or nonmyristoylated Nef (TH) were used to monitor the expression and activation of 10 PKC isoforms. The same clones were used to evaluate the effect of Nef on the viral long terminal repeat (LTR) promoter after activation of PKC with the phorbol ester 12-myristate 13-acetate (PMA). PKC intracellular distribution and activation were evaluated by Western blot analysis of cytosolic and membrane fractions of control and Nef-expressing clones. PMA-induced LTR activation was analyzed in clones transfected with a plasmid encoding for the CAT reporter gene controlled by the LTR promoter, by using an enzyme-linked immunosorbent assay to measure CAT expression. Nef selectively downregulated the expression and activation of betaII and epsilon PKC isoforms in astrocytoma cells. Such downregulation correlated with an inhibition of LTR activation after PMA stimulation. The myristoylation of Nef and its membrane localization were essential for these effects. These results suggest that Nef may alter astrocytic functions by interfering with PKC expression and activation and contribute to the restriction of HIV-1 replication in astrocytes.


The neuroendocrine precursor 7B2 is a sulfated protein proteolytically processed by a ubiquitous furin-like convertase.
The neuroendocrine granule-associated protein 7B2, unlike many other neuroendocrine precursor proteins stored in secretory granules, carries in its primary structure the Arg-Xaa-Arg/Lys-Arg processing site usually found in constitutively secreted precursor proteins and recognized by the ubiquitously expressed convertase, furin. pro7B2 (30 kDa), when expressed in endocrine (AtT-20, PC12, and GH4C1) or non-endocrine (Ltk-) cell lines using recombinant vaccinia viruses, was converted to a 23-kDa form. Mutation of the P4 Arg to Gly completely prevented this conversion. When excess pro7B2 was coexpressed with the pro-protein convertases PC1, PC2, or furin, only furin could induce complete processing. In addition, coexpression of pro7B2 in LoVo cells, which are devoid of endogenous furin activity, with each one of the three convertases, showed that only furin was able to induce processing of this precursor. pro7B2 processing in AtT-20 was completely abolished when protein transport into Golgi compartments was blocked by cell incubation at either 15 or 37 degrees C in the presence of monensin or brefeldin A. Furthermore, pulse-chase experiments in the presence of Na2[35S]SO4 showed that pro7B2 is Tyr-sulfated in the trans-Golgi network before it is processed. These results demonstrate that pro7B2 is first processed by a furin-like enzyme within the trans-Golgi network into a 23-kDa form that is then sequestered into secretory granules.


Transport and assembly of gag proteins into Moloney murine leukemia virus.
We have studied the process of Moloney murine leukemia virus (M-MuLV) assembly by characterization of core (gag) protein mutants and analysis of wild-type (wt) gag proteins produced by cells in the presence of the ionophore monensin. Our genetic studies involved examination of linker insertion mutants of a Gag-beta-galactosidase (Gag-beta-gal) fusion protein, GBG2051, which is incorporated into virus particles when expressed in the presence of wt viral proteins. Analysis indicated that the amino-terminal two-thirds of the gag matrix domain is essential for targeting of proteins to the plasma membrane; mutant proteins localized to the cytoplasm or were trapped on intracellular membranes. Mutations through most of the coding region of the gag capsid domain generated proteins which were released from cells in membrane vesicles but not in virions. In contrast, linker insertions into p12gag or carboxy-terminal portions of the matrix or capsid coding regions did not affect assembly of fusion proteins into virus particles. Monensin, which blocks vesicular transport, inhibited gag protein intracellular transport and release from cells. Our results suggest that a significant proportion of M-MuLV myristylated gag proteins travel via vesicles to the cell surface. Specific matrix protein polypeptide regions and myristic acid modification are both necessary for appropriate gag protein transport, while capsid protein interactions appear to mediate the final phase of virion formation.


Akr1p and the type I casein kinases act prior to the ubiquitination step of yeast endocytosis: Akr1p is required for kinase localization to the plasma membrane.
Ubiquitination of the plasma membrane-localized yeast a-factor receptor (Ste3p) triggers a rapid, ligand-independent endocytosis leading to its vacuolar degradation. This report identifies two mutants that block uptake by blocking ubiquitination, these being mutant either for the ankyrin repeat protein Akr1p or for the redundant type I casein kinases Yck1p and Yck2p. While no obvious defect was seen for wild-type Ste3p phosphorylation in akr1 or yck mutant backgrounds, examination of the Delta320-413 Ste3p deletion mutant phosphorylation did reveal a clear defect in both mutants. The Delta320-413 deletion removes 18 Ser-Thr residues (possible YCK-independent phosphorylation sites) yet retains the 15 Ser-Thr residues of the Ste3p PEST-like ubiquitination-endocytosis signal. Two other phenotypes link akr1 and yck mutants: both are defective in phosphorylation of wild-type alpha-factor receptor, and while both are defective for Ste3p constitutive internalization, both remain partially competent for the Ste3p ligand-dependent uptake mode. Yck1p-Yck2p may be the function responsible in phosphorylation of the PEST-like ubiquitination-endocytosis signal. Akr1p appears to function in localizing Yck1p-Yck2p to the plasma membrane, a localization that depends on prenylation of C-terminal dicysteinyl motifs. In akr1Delta cells, Yck2p is mislocalized, showing a diffuse cytoplasmic localization identical to that seen for a Yck2p mutant that lacks the C-terminal Cys-Cys, indicating a likely Akr1p requirement for the lipid modification of Yck2p, for prenylation, or possibly for palmitoylation.


Developmental changes in the modification of lysosomal enzymes in Dictyostelium discoideum.
Evidence has been found for a generalized change in the post-translational modification of lysosomal enzymes during development of Dictyostelium discoideum. The physical and antigenic properties of four developmentally regulated lysosomal enzymes, N-acetylglucosaminidase, beta-glucosidase, alpha-mannosidase, and acid phosphatase, have been examined throughout the life cycle. In vegetative cells, a single major isoelectric species is detected for each enzymatic activity on native nonequilibrium isoelectric focusing gels. Between 6 and 10 hr of development, all activities, including the preformed enzyme, become less negatively charged, resulting in a modest but reproducible shift in the isoelectric focusing pattern. This alteration is not detected by native gel electrophoresis at constant pH. As development continues, the specific activity of beta-glucosidase, alpha-mannosidase, and acid phosphatase continues to increase and coincidentally, new, less acidic isozymic bands of activity can be observed on both gel systems. Some of these new isozymes accumulate preferentially in anterior cells, while others accumulate preferentially in posterior cells of migrating slugs. N-Acetylglucosaminidase does not increase in specific activity late in development and no new isozymic species appear. Using a monoclonal antibody that reacts with sulfated N-linked oligosaccharides shared by vegetative lysosomal enzymes in D. discoideum, the antigenicity of the developmental isozymes has been characterized. All of the enzymatic activity present during vegetative growth and early development is immunoprecipitable. However, the less negatively charged isozymes that accumulate after aggregation are not recognized by the antibody. Nonantigenic acid phosphatase and alpha-mannosidase are found in both anterior and posterior cells from migrating pseudoplasmodia. Since each enzyme is coded by a single structural gene, these results suggest that the isozymes present late in development arise from the synthesis of the same polypeptides with altered post-translational modifications. The appearance of anterior and posterior specific isozymes is likely to be the result of cell type specific changes in the glycoprotein modification pathway for newly synthesized proteins.


Involvement of Src family protein tyrosine kinases in Ca(2+) sensitization of coronary artery contraction mediated by a sphingosylphosphorylcholine-Rho-kinase pathway.
We recently reported that sphingosylphosphorylcholine (SPC) is a novel messenger for Rho-kinase-mediated Ca(2+) sensitization of vascular smooth muscle (VSM) contraction. Subcellular localization and kinase activity of Src family protein kinases (SrcPTKs), except for c-Src, is controlled by a reversible S-palmitoylation, an event inhibited by eicosapentaenoic acid (EPA). We examined the possible involvement of SrcPTKs in SPC-induced Ca(2+) sensitization and effects of EPA. We used porcine coronary VSM and rat aortic VSM cells (VSMCs) in primary culture. An SrcPTKs inhibitor, PP1, and EPA inhibited SPC-induced contraction, concentration-dependently, without affecting [Ca(2+)](i) levels and the Ca(2+)-dependent contraction induced by high K(+) depolarization. A digitized immunocytochemical analysis in VSMCs revealed that SPC induced translocation of Fyn, but not of c-Src, from the cytosol to the cell membrane, an event abolished by EPA. Translocation of Rho-kinase from the cytosol to the cell membrane by SPC was also inhibited by EPA and PP1. The SPC-induced activation of SrcPTKs was blocked by EPA and PP1, but not by Y27632, an Rho-kinase inhibitor. Rho-kinase-dependent phosphorylation of myosin phosphatase induced by SPC was inhibited by EPA, PP1, and Y27632. Translocation and activation of SrcPTKs, including Fyn, play an important role in Ca(2+) sensitization of VSM contractions mediated by a SPC-Rho-kinase pathway.


Chemical characterization of the parasitophorous vacuole membrane antigen QF 116 from Plasmodium falciparum.
On the basis of amino acid sequencing and immunological cross-reactivity, the Plasmodium falciparum parasitophorous vacuole antigens QF116 and exp-1/CRA are apparently identical. The epitope recognized by an inhibitory monoclonal antibody directed against QF116 is located proximal to the C-terminus of the protein. The QF116 protein is processed during maturation by the cleavage of a 22-amino-acid signal peptide and acylated as measured by labeling with myristic acid.


Properties of type II inositol polyphosphate 5-phosphatase.
We have isolated additional cDNA clones encoding type II inositol polyphosphate 5-phosphatase (5-phosphatase II) resulting in a combined cDNA of 3076 nucleotides encoding a protein of 942 amino acids. The 5-phosphatase II hydrolyzed both Ins(1,4,5)P3 to Ins(1,4)P2 and the phospholipid PtdIns(4,5)P2 to PtdIns(4)P both in vitro and in vivo. There are two motifs highly conserved between types I and II 5-phosphatase and several other proteins presumed to be inositol phosphatases suggesting a possible role in catalysis. The type II 5-phosphatase also contains homology to several GTPase activating proteins although no such activity for 5-phosphatase II was found. The predicted protein ends with the sequence CNPL, suggesting that it is isoprenylated as a mechanism for membrane attachment. We found evidence for isoprenylation by demonstrating incorporation of [3H]mevalonate into native but not C939S mutant 5-phosphatase II expressed in Sf9 insect cells. Furthermore, we showed that membrane localization and the activity of 5-phosphatase II toward its lipid substrate PtdIns(4,5)P2 is reduced by eliminating 5-phosphatase II isoprenylation in the mutant C939S relative to the native enzyme.


Terminal marking of avian triosephosphate isomerases by deamidation and oxidation.
Triosephosphate isomerase (TPI) provides an excellent model for terminal marking and protein degradation. Mammalian TPI is terminally modified by deamidation at Asn71-Gly, resulting in unfolding, dissociation, and proteolysis. In contrast, chicken TPI, which contains a lysine at position 71, is terminally modified by the oxidation of Cys126. Thus, both deamidation and oxidation initiate degradation of TPI from different species. To explore the terminal marking in other avians, we have purified the turkey TPI to homogeneity and determined its characteristics. Although the molecular properties of the turkey and chicken TPI were very similar, their tolerances to temperature, oxidants, and alkaline pH were very different. For example, chicken TPI was inactivated 80% in either 10 mM oxidized glutathione or H2O2, whereas 120 mM GSSG had no effect on turkey TPI, and > 120 mM H2O2 was needed for 80% inactivation. Under alkaline conditions that cause rapid deamidation of the mammalian TPI, neither avian enzyme deamidated. Chicken TPI, however, aggregated. Aggregation was reversed by 2-mercaptoethanol. Under prolonged exposure to milder conditions the turkey enzyme was completely inactivated and deamidated at Asn15-Gly. Thus, there are marked differences in the susceptibility of these two avian enzymes to oxidation and deamidation, and their terminal marking mechanisms appear to be different.


Simple histone acetylation plays a complex role in the regulation of gene expression.
Eukaryotic DNA is packaged into chromatin by histone proteins, which assemble the DNA into an organized, higher-order structure. The precise organization of chromatin is essential for faithful execution of DNA-mediated reactions such as transcription, DNA replication, DNA repair and DNA recombination. The organization of chromatin is considered to be regulated by a variety of post-translational modifications of histones, such as acetylation, methylation, phosphorylation, ubiquitination, SUMOylation and poly-ADP-ribosylation. The relationship between histone acetylation and gene expression was first observed in 1964. Since then, a great deal of evidence has accumulated showing that not only transcription but other DNA-mediated reactions also are regulated by histone acetylation. With regard to the putative mechanism(s) by which histone acetylation regulates the flow of genetic information, site-specific modification and recognition of acetylated histone/DNA complexes have been postulated. Elucidation of the downstream effects of histone modification, as well as the identification, isolation and characterization of the relevant factors involved, have aided in our understanding of the mechanisms of regulation of DNA activity by histones. Currently, state-of-the-art technologies that enable genome-wide analysis are allowing insight into a critical and interesting question in eukaryotic transcription: are the principles that govern transcription of individual gene loci applicable to the genome as a whole? Here, we review the recent progress on histone modifications, with an emphasis on the role of histone acetylation in gene expression.


Characterization of an acyltransferase acting on p21N-ras protein in a cell-free system.
We have identified a protein-acyltransferase activity in membranes from mouse fibroblasts which transfers palmitate from palmitoyl-CoA to p21N-ras. Specificity of acylation has been confirmed by linkage analysis using hydroxylamine and by peptide mapping of in vivo and in vitro acylated p21N-ras. The acylation was temperature- and time-dependent, and prevented by prior boiling of membranes, consistent with an enzymatic process. The activity was detected in membranes, but not cytosol, and co-fractionated on Percoll gradients with Golgi markers. Cytosolic p21N-ras from mouse fibroblasts, which is C-terminally modified at its CAAX sequence, was a better substrate for the enzyme that recombinant bacterially expressed, unmodified p21N-ras. The activity could be solubilised in non-ionic detergents, making it amenable to purification.


Increased acylated plasma ghrelin, but improved lipid profiles 24-h after consumption of carob pulp preparation rich in dietary fibre and polyphenols.
We have recently shown that a polyphenol-rich insoluble dietary fibre preparation from carob pulp (Ceratonia siliqua L; carob fibre) decreased postprandial acylated ghrelin, TAG and NEFA during an acute liquid meal challenge test. However, delayed effects of carob fibre consumption are unknown. Therefore, a randomized controlled crossover study in nineteen healthy volunteers consuming foods with or without 50 g carob fibre was conducted. On the subsequent day (day 2), glucose, TAG, total and acylated ghrelin as well as insulin, NEFA and leptin were assessed at baseline and at timed intervals for 300 min after ingestion of standardized bread. Consumption of carob fibre-enriched foods did not affect fasting concentrations of glucose, TAG, total ghrelin, NEFA, insulin and leptin. Fasting acylated ghrelin was increased on the day subsequent to carob fibre consumption compared with control (P = 0.046). After consumption of the standard bread on day 2, glucose response (P = 0.029) was increased, and TAG (P = 0.033) and NEFA (P < 0.001) responses were decreased compared with control. Postprandial responses of total and acylated ghrelin, insulin and leptin on day 2 were unaffected by carob fibre consumption the previous day. In conclusion, an increase in total and acylated plasma ghrelin accompanied by enhanced lipid metabolism after carob fibre consumption suggests higher lipid utilization and suppressed lipolysis on the day subsequent to carob fibre consumption. However, elevated glucose levels after carob fibre consumption need to be addressed in future studies.


Multiple palmitoylation of synaptotagmin and the t-SNARE SNAP-25.
Synaptotagmin, a likely calcium sensor for synaptic transmission, and SNAP-25, a t-SNARE of the presynaptic plasma membrane, are key proteins for the docking and fusion of synaptic and other vesicles. We report that both synaptotagmin and SNAP-25 are palmitoylated with their fatty acids attached in a labile thioester-type bond. A SNAP-25 mutant with deleted palmitoylation sites is found exclusively in the cytosol after cell fractionation, whereas the palmitoylated form of SNAP-25 is membrane-bound, establishing that SNAP-25 is membrane-anchored via covalently linked palmitate.


Shift of serum osteocalcin components between cord blood and blood at day 5 of life.
Vitamin K deficiency is a relatively common condition in neonates. However, the role of vitamin K in neonatal bone metabolism remains to be determined. Osteocalcin (OC) is the most abundant noncollagenous protein in bone, and is regulated to be gamma-carboxylated by vitamin K. In this study, we measured gamma-carboxylated osteocalcin (Gla-OC) and non- or undercarboxylated osteocalcin (Glu-OC) separately, and examined the effects of vitamin K on osteocalcin metabolism. Eighteen full-term healthy neonates were enrolled in this study. In the cord and d-5 blood samples, the OC levels were determined by three different methods to examine the intact OC by immunoradiometric assay (IRMA), Gla-OC, and Glu-OC. Serum vitamin K fractions, hepaplastin test, and type 1 procollagen carboxyl extension peptide were also determined. Urine samples were also collected from the first voiding and on d 5 to determine urinary pyridinoline, deoxypyridinoline, and gamma-carboxylated glutamic acid. Serum levels of phylloquinone (PK) and menaquinone (MK)-4 increased on d 5 following vitamin K administration and increased intake in breast milk and/or formula. The OC levels determined by IRMA did not change between cord and d-5 blood samples, but the Gla-OC level increased remarkably and Glu-OC reduced to a negligible level. OC in cord blood is mainly Glu-OC, and Glu-OC is replaced with Gla-OC within 5 d of life after vitamin K supplement. The IRMA assay fails to distinguish Gla-OC from Glu-OC and caution is needed to estimate bone turnover with this method in the perinatal period.


A dominant-negative UBC12 mutant sequesters NEDD8 and inhibits NEDD8 conjugation in vivo.
NEDD8, a novel ubiquitin-like protein, has been shown to conjugate to proteins in a manner analogous to ubiquitination and sentrinization. Recently, human UBC12 was identified as a putative NEDD8 conjugation enzyme (E2). While investigating the in vivo function of UBC12, we found that the point mutant, UBC12(C111S), showed a dominant-negative effect on NEDD8 conjugation. This mutant, with a single Cys-to-Ser substitution at the conserved Cys residue in the E2 family, could specifically inhibit NEDD8 conjugation. We observed the dominant-negative effect on NEDD8 conjugation to substrates, including the C-terminal fragment of cullin-2 (Cul-2-DeltaN), full-length cullin-1, and also other uncharacterized target proteins. Interestingly, UBC12(C111S) formed a heterodimeric conjugate with NEDD8. This conjugate was stable under stringent conditions, including 6 m guanidine HCl, 8 m urea, 2% SDS, or 5% beta-mercaptoethanol. Our results are consistent with the hypothesis that UBC12(C111S) sequesters the NEDD8 monomer by forming a UBC12(C111S)-NEDD8 conjugate and, in turn, inhibits the subsequent transfer of NEDD8 to its targets. To examine the biological role of NEDD8 conjugation, this dominant-negative form of UBC12 was applied to a cell growth assay. Overexpression of UBC12(C111S) led to inhibition of growth in U2OS and HEK293 cells. Thus, this dominant-negative form of UBC12 could be useful in defining the role of NEDD8 modification in other biological systems.


The redox domain of the Yap1p transcription factor contains two disulfide bonds.
The subcellular localization of the Saccharomyces cerevisiae transcription factor Yap1p is regulated by oxidation and reduction. We purified Yap1p from yeast and characterized its properties in vitro. Electrophoretic mobility shift assays showed that the purified protein can specifically bind the TRX2 target promoter. Yap1p was purified under reducing conditions, but removal of reducing agents resulted in the formation of an oxidized Yap1p species with properties similar to in vivo oxidized Yap1p. MALDI-TOF mass spectrometry analysis revealed that the oxidized form of Yap1p contains two disulfide bonds between C303-C598 and C310-C629. A stable domain of approximately 15 kDa was detected upon limited proteolysis of oxidized but not reduced Yap1p. This Yap1p protease resistant domain was purified, and MALDI-TOF mass spectrometry analysis showed that it was comprised of two separate cysteine-containing peptides of Yap1p. These peptides are separated by 250 amino acids and are joined by the C303-C598 and C310-C629 disulfide bonds. Taken together, these data suggest that the domain that controls Yap1p subcellular localization is modular and contains a redox center comprised of four cysteine residues.


Long-term alcohol effects on hepatic phosphoglucomutase activities in relation to posttranslational modification of the protein.
ADP-ribosylation is a posttranslational protein modification catalyzed by two classes of enzymes: mono-ADP-ribosyltransferase and poly-ADP-ribose polymerases. We previously demonstrated that long-term alcohol intake remarkably enhanced an endogenous ADP-ribosylation of a 58 kDa protein in rat liver and also identified the 58 kDa protein as phosphoglucomutase (PGM). To assess biological significance of this phenomenon, we tested the effects of long-term alcohol intake on PGM activities in connection with posttranslational modification of the protein. ADP-ribosylation of PGM was mono- rather than poly-ADP-ribosylation. Also, nonenzymatic binding of ADP-ribose was excluded. It was of note that ADP-ribosylation of exogenous PGM was remarkably increased by adding rat liver plasma membranes, and that the extent of the increase was greater in alcohol-fed rats than in pair-fed controls. Furthermore, PGM activities were significantly increased after long-term alcohol intake concomitant with increased ADP-ribosyltransferase activities toward PGM. In view of the variety of roles of PGM in the liver, such as carbohydrate metabolism and Ca2+ homeostasis, it is tempting to speculate that increased ADP-ribosylation of PGM may play a role in long-term alcohol effects on hepatocytes.


Identification of amino acids in the gamma-carboxylation recognition site on the propeptide of prothrombin.
A gamma-carboxylation recognition site on the propeptide of the vitamin K-dependent blood coagulation proteins directs the carboxylation of glutamic acid residues by binding to the vitamin K-dependent carboxylase. To determine residues that define this site, we evaluated the effect of mutation of certain residues in the prothrombin propeptide on the extent of carboxylation. The prothrombin cDNA modified by site-specific mutagenesis was expressed in Chinese hamster ovary cells using a system that yields functional fully carboxylated prothrombin. The cell supernatants containing recombinant prothrombin were evaluated for the extent of gamma-carboxylation by immunoassay. Conformation-specific anti-prothrombin:Ca(II)-specific antibodies measure native completely carboxylated prothrombin; anti-prothrombin:total antibodies measure all forms of prothrombin, regardless of gamma-carboxyglutamic acid content. Mutation of His-18 to Gly, Val-17 to Ser, Leu-15 to Gly or Asp, or Ala-10 to Asp was associated with a partial (30-65%) inhibition of gamma-carboxylation. Mutation of Ala-14 to Ser or Ser-8 to Val did not inhibit gamma-carboxylation. From this and earlier work, residues whose mutation leads to a significant impairment of carboxylation include His-18, Val-17, Phe-16, Leu-15, and Ala-10. Residues whose mutation does not alter the carboxylation recognition site include Ala-14, Ser-8, Arg-4, and Arg-1. To determine the size of the recognition site, the in vitro carboxylation of propeptide-containing synthetic peptides was compared. A 28-residue peptide, based upon residues -18 to +10 of prothrombin, and a 54-residue peptide, based upon residues -18 to +36 of prothrombin, were carboxylated by partially purified bovine carboxylase with similar Km values of 2-5 microM. These results indicate that the gamma-carboxyglutamic acid-rich region of prothrombin makes a minimal contribution to carboxylase binding. A molecular surface of about five amino acids located within the propeptide appears to define the carboxylation recognition site on the precursor forms of the vitamin K-dependent proteins.


The lipoatrophic caveolin-1 deficient mouse model reveals autophagy in mature adipocytes.
Adipose tissue lipoatrophy caused by caveolin gene deletion in mice is not linked to defective adipocyte differentiation. We show that adipose tissue development cannot be rescued by endothelial specific caveolin-1 re-expression, indicating primordial role of caveolin in mature adipocytes. Partial or total caveolin deficiency in adipocytes induced broad protein expression defects, including but not limited to previously described downregulation of insulin receptor. Global alterations in protein turnover, and accelerated degradation of long-lived proteins were found in caveolin-deficient adipocytes. Lipidation of endogenous LC3 autophagy marker and distribution of GFP-LC3 into aggregates demonstrated activated autophagy in the absence of caveolin-1 in adipocytes. Furthermore, electron microscopy revealed autophagic vacuoles in caveolin-1 deficient but not control adipocytes. Surprisingly, significant levels of lipidated LC3-II were found around lipid droplets of normal adipocytes, maintained in nutrient-rich conditions or isolated from fed mice, which do not display autophagy. Altogether, these data indicate that caveolin deficiency induce autophagy in adipocytes, a feature that is not a physiological response to fasting in normal fat cells. This likely resulted from defective insulin and lipolytic responses that converge in chronic nutrient shortage in adipocytes lacking caveolin-1. This is the first report of a pathological situation with autophagy as an adaptative response to adipocyte failure.


Posttranslational processing of human alpha 2-HS glycoprotein (human fetuin). Evidence for the production of a phosphorylated single-chain form by hepatoma cells.
alpha 2-HS glycoprotein (alpha 2-HS) is a major protein occurring in human blood and calciferous tissues. Due to extensive sequence identity, alpha 2-HS has been grouped with the fetuins, a family of proteins that occur in fetal plasma in high concentrations. Native alpha 2-HS undergoes a series of posttranslational modifications including proteolytic processing, multiple N-glycosylations and O-glycosylations, and sulfation of the carbohydrate side chains. Various two-chain forms of alpha 2-HS have been prepared from human plasma, however, the single-chain precursor has not yet been isolated. Here, we have studied the biosynthesis of alpha 2-HS by a human hepatoma cell line, HepG2. We demonstrate that a single-chain form and the two-chain form of alpha 2-HS are secreted by this cell line. The alpha 2-HS forms are further modified by phosphorylation on multiple serine residues. Mapping studies indicate that the connecting peptide region releasable from the heavy chain of alpha 2-HS contains at least one such phosphorylation site. Our results identify proteolytic trimming and/or phosphorylation as modifications possibly regulating the biological effects of alpha 2-HS and the homologous fetuins.


One SUMO is sufficient to silence the dimeric potassium channel K2P1.
Small ubiquitin modifier 1 (SUMO1) is shown to regulate K2P1 background channels in the plasma membrane (PM) of live mammalian cells. Confocal microscopy reveals native SUMO1, SAE1, and Ubc9 (the enzymes that activate and conjugate SUMO1) at PM where SUMO1 and expressed human K2P1 are demonstrated to colocalize. Silent K2P1 channels in excised PM patches are activated by SUMO isopeptidase (SENP1) and resilenced by SUMO1. K2P1-Lys274 is crucial: when mutated to Gln, Arg, Glu, Asp, Cys, or Ala, the channels are constitutively active and insensitive to SUMO1 and SENP1. Tandem mass spectrometry confirms conjugation of SUMO1 to the epsilon-amino group of Lys274 in vitro. FRET microscopy shows that assembly of K2P1 and SUMO1 requires Lys274. Single-particle TIRF microscopy shows that wild-type channels in PM have two K2P1 subunits and assemble with two SUMO1 monomers. Although channels engineered with one Lys274 site carry just one SUMO1 they are activated and silenced by SENP1 and SUMO1 like wild-type channels.


Phosphorylation of VACM-1/Cul5 by protein kinase A regulates its neddylation and antiproliferative effect.
Expression of the VACM-1/cul5 gene in endothelial and in cancer cell lines in vitro inhibits cellular proliferation and decreases phosphorylation of MAPK. Structure-function analysis of the VACM-1 protein sequence identified consensus sites specific for phosphorylation by protein kinases A and C (PKA and PKC) and a Nedd8 protein modification site. Mutations at the PKA-specific site in VACM-1/Cul5 ((S730A)VACM-1) sequence resulted in increased cellular growth and the appearance of a Nedd8-modified VACM-1/Cul5. The aim of this study was to examine if PKA-dependent phosphorylation of VACM-1/Cul5 controls its neddylation status, phosphorylation by PKC, and ultimately growth. Our results indicate that in vitro transfection of rat adrenal medullary endothelial cells with anti-VACM-1-specific small interfering RNA oligonucleotides decreases endogenous VACM-1 protein concentration and increases cell growth. Western blot analysis of cell lysates immunoprecipitated with an antibody directed against a PKA-specific phosphorylation site and probed with anti-VACM-1-specific antibody showed that PKA-dependent phosphorylation of VACM-1 protein was decreased in cells transfected with (S730A)VACM-1 cDNA when compared with the cytomegalovirus-transfected cells. This change was associated with increased modification of VACM-1 protein by Nedd8. Induction of PKA activity with forskolin reduced modification of VACM-1 protein by Nedd8. Finally, rat adrenal medullary endothelial cells transfected with (S730A)VACM-1/cul5 cDNA and treated with phorbol 12-myristate 13-acetate (10 and 100 nm) to induce PKC activity grew significantly faster than the control cells. These results suggest that the antiproliferative effect of VACM-1/Cul5 is dependent on its posttranslational modifications and will help in the design of new anticancer therapeutics that target the Nedd8 pathway.


Molecular cloning and characterization of an isoprenylated 67 kDa protein.
The cDNA coding for a 67 kDa protein (p67) was isolated from a rat Schwann cell library. A recombinant form of p67 expressed in bacteria was used to produce polyclonal anti-p67 antibodies. By immunoblot analysis p67 was found to be expressed in most tissues and cell lines examined. Inspection of the deduced amino acid sequence revealed a COOH-terminal consensus sequence for isoprenylation. Consistent with this finding, p67 was a substrate for isoprenylation in vitro by geranylgeranylpyrophosphate. p67 was associated predominantly with the particulate fraction of rat smooth muscle cells. The rat p67 sequence was highly homologous to a family of recently described human and mouse gamma-interferon inducible, guanine nucleotide binding proteins.


Not insulin but insulin sensitivity, leptin, and cortisol are major factors regulating serum acylated ghrelin level in healthy women.
It has been suggested that insulin and glucose are the most important factors for ghrelin secretion. Most of these studies were performed using total ghrelin assays, detecting two forms of ghrelin (acylated and desacyl), derived from the same peptide precursor but having different biological effects. This study was therefore designed to characterize associations between serum acylated ghrelin levels (Ghr), selected adipocytokines, hormones, and carbohydrate metabolism parameters in healthy women in stable energy metabolism. The study was performed on 32 healthy, normal-weight, non-pregnant women with normal [body mass index (BMI) 18.9-24.2 kg/m2] and stable (the difference between two measurements performed within 1 month being less than 0.5 kg) body weight, aged 22-47 yr. Leptin, Ghr, GH, IGF-I, cortisol, insulin, and glucose were measured in the early follicular phase of the menstruation cycle. Insulin sensitivity was measured using quantitative insulin sensitivity check index (QUICKI). Body composition was assessed by bioimpedance. We found a positive linear correlation between leptin and Ghr (r=0.375; p=0.034) and negative correlation between insulin and Ghr (r=-0.374; p=0.034). GH, IGF-I, adiponectin, and body composition parameters did not correlate with Ghr. In multiple regression analysis only QUICKI, leptin, glucose, and cortisol (positively) and age (negatively) accounted for 50% variation of Ghr. Insulin and BMI did not contribute significantly to the model. Our results suggest that in healthy women basal Ghr level is regulated by multiple factors, mainly by insulin sensitivity, leptin, and adrenal glands activity. However, further studies are needed to elucidate the physiological mechanisms involved in acylated Ghr secretion.


Atg21 is a phosphoinositide binding protein required for efficient lipidation and localization of Atg8 during uptake of aminopeptidase I by selective autophagy.
Delivery of proteins and organelles to the vacuole by autophagy and the cytoplasm to vacuole targeting (Cvt) pathway involves novel rearrangements of membrane resulting in the formation of vesicles that fuse with the vacuole. The mechanism of vesicle formation and the origin of the membrane are complex issues still to be resolved. Atg18 and Atg21 are proteins essential to vesicle formation and together with Ygr223c form a novel family of phosphoinositide binding proteins that are associated with the vacuole and perivacuolar structures. Their localization requires the activity of Vps34, suggesting that phosphatidylinositol(3)phosphate may be essential for their function. The activity of Atg18 is vital for all forms of autophagy, whereas Atg21 is required for the Cvt pathway but not for nitrogen starvation-induced autophagy. The loss of Atg21 results in the absence of Atg8 from the pre-autophagosomal structure (PAS), which may be ascribed to a reduced rate of conjugation of Atg8 to phosphatidylethanolamine. A similar defect in localization of a second ubiquitin-like conjugate, Atg12-Atg5, suggests that Atg21 may be involved in the recruitment of membrane to the PAS.


S-palmitoylation modulates estrogen receptor alpha localization and functions.
17beta-Estradiol (E2) acts as a chemical messenger in target tissues inducing both slow nuclear and rapid extra-nuclear responses. E2 binds to its cognate nuclear receptors (ER) resulting in the activation of target gene transcription in the nucleus. In addition to these genomic effects, E2 modulates cell functions through rapid non-genomic actions. Stimulation of G-proteins, Ca(2+) influx, inositol phosphate generation as well as phospholipase C, ERK/MAPK, and PI3K/AKT activation all occur within seconds to minutes after E2 binding to a small population of ERalpha located at the plasma membrane. The great impact of these rapid signals on cell physiology renders central the knowledge of the structural bases and mechanisms that mediate extra-nuclear signaling by E2. Several laboratories, including our own, have recently elucidated the structural requirements for localization and function of plasma membrane ERalpha. This review summarizes the molecular mechanisms of E2-induced rapid non-genomic actions relevant for cell functions, highlighting the role of lipid modification (i.e., palmitoylation) in the ERalpha localization to and residence at the plasma membrane.


Characterization of subcellular localization of duck enteritis virus UL51 protein.
BACKGROUND: Knowledge of the subcellular localization of a protein can provide useful insights about its function. While the subcellular localization of many alphaherpesvirus UL51 proteins has been well characterized, little is known about where duck enteritis virus (DEV) UL51 protein (pUL51) is targeted to. Thus, in this study, we investigated the subcellular localization and distribution of DEV pUL51 by computer aided analysis, as well as indirect immunofluorescence (IIF) and transmission immunoelectron microscopy (TIEM) approaches in DEV-infected cells. RESULTS: The DEV UL51 gene product was identified as an approximate 34 kDa protein in DEV-infected cells analyzed by western blotting. Computer aided analysis suggested that DEV pUL51 is not targeted to the mitochondrial, extra-cellular or nucleus, but be targeted to the cytoplasmic in host cells, more specifically, palmitoylation of the pUL51 through the N-terminal cysteine at position 9 makes membrane association and Golgi localization possible. Using IIF analysis, we found that DEV pUL51 was first detected in a juxtanuclear region of DEV-infected cells at 9 h postinfection (p.i.), and then was detected widely distributed in the cytoplasm and especially was stronger in the juxtanuclear region from 12 to 60 h p.i. TIEM analysis revealed that DEV pUL51 was mainly associated with cytoplasmic virions and also with some membranous structure near the pUL51-specific immuno-labeling intracellular virion in the cytoplasmic vesicles; moreover, the pUL51 efficiently accumulated in the Golgi apparatus at first, and then was sent to the plasma membrane from the Golgi by some unknown mechanism. CONCLUSION: In this work, we described the basic characteristics of pUL51 subcellular localization and distribution for the first time. From these results, we concluded that palmitoylation at the N-terminal cysteine, which is conserved in all alphaherpesvirus UL51 homologs, is required for its membrane association and Golgi localization, and the pUL51 mainly localized to the juxtanuclear region of DEV-infected cells, as well seemed to be incorporated into mature virions as a component of the tegument. The research will provide useful clues for DEV pUL51 functional analysis, and will be usefull for further understanding the localization properties of alphaherpesvirus UL51 homologs.


Prenylated Rab acceptor protein is a receptor for prenylated small GTPases.
Localization of Ras and Ras-like proteins to the correct subcellular compartment is essential for these proteins to mediate their biological effects. Many members of the Ras superfamily (Ha-Ras, N-Ras, TC21, and RhoA) are prenylated in the cytoplasm and then transit through the endomembrane system on their way to the plasma membrane. The proteins that aid in the trafficking of the small GTPases have not been well characterized. We report here that prenylated Rab acceptor protein (PRA1), which others previously identified as a prenylation-dependent receptor for Rab proteins, also interacts with Ha-Ras, RhoA, TC21, and Rap1a. The interaction of these small GTPases with PRA1 requires their post-translational modification by prenylation. The prenylation-dependent association of PRA1 with multiple GTPases is conserved in evolution; the yeast PRA1 protein associates with both Ha-Ras and RhoA. Earlier studies reported the presence of PRA1 in the Golgi, and we show here that PRA1 co-localizes with Ha-Ras and RhoA in the Golgi compartment. We suggest that PRA1 acts as an escort protein for small GTPases by binding to the hydrophobic isoprenoid moieties of the small GTPases and facilitates their trafficking through the endomembrane system.


Trafficking and postsecretory events responsible for the formation of secreted human salivary peptides: a proteomics approach.
To elucidate the localization of post-translational modifications of different classes of human salivary proteins and peptides (acidic and basic proline-rich proteins (PRPs), Histatins, Statherin, P-B peptide, and "S type" Cystatins) a comparative reversed phase HPLC-ESI-MS analysis on intact proteins of enriched granule preparations from parotid and submandibular glands as well as parotid, submandibular/sublingual (Sm/Sl), and whole saliva was performed. The main results of this study indicate the following. (i) Phosphorylation of all salivary peptides, sulfation of Histatin 1, proteolytic cleavages of acidic and precursor basic PRPs occur before granule storage. (ii) In agreement with previous studies, basic PRPs are secreted by the parotid gland only, whereas all isoforms of acidic PRPs (aPRPs) are secreted by both parotid and Sm/Sl glands. (iii) Phosphorylation levels of aPRPs, Histatin 1, and Statherin are higher in the parotid gland, whereas the extent of cleavage of aPRP is higher in Sm/Sl glands. (iv) O-Sulfation of tyrosines of Histatin 1 is a post-translational modification specific for the submandibular gland. (v) The concentration of Histatin 3, Histatin 5, and Histatin 6, but not Histatin 1, is higher in parotid saliva. (vi) Histatin 3 is submitted to the first proteolytic cleavage (generating Histatins 6 and 5) during granule maturation, and it occurs to the same relative extent in both glands. (vii) The proteolytic cleavages of Histatin 5 and 6, generating a cascade of Histatin 3 fragments, take place after granule secretion and are more extensive in parotid secretion. (viii) Basic PRPs are cleaved in the oral cavity by unknown peptidases, generating various small proline-rich peptides. (ix) C-terminal removal from Statherin is more extensive in parotid saliva. (x) P-B peptide is secreted by both glands, and its relative quantity is higher in submandibular/sublingual secretion. (xi) In agreement with previous studies, S type Cystatins are mainly the product of Sm/Sl glands.


NUB1 promotes cytoplasmic localization of p53 through cooperation of the NEDD8 and ubiquitin pathways.
Non-covalent recognition of ubiquitin (Ub) and ubiquitin-like molecules (Ubls) by interacting proteins has an important role in the regulation of protein function and initiation of signalling events. In addition, growing evidence suggests that regulation of p53 subcellular localization contributes to the biological outcome of the p53 response. Cytoplasmic p53 is shown to promote apoptosis and inhibit the induction of autophagy. In this study we show that NEDD8 ultimate buster 1 (NUB1), a non-covalent interactor of the Ubl NEDD8 (neural precursor cell expressed, developmentally downregulated 8), controls the localization of p53. Expression of NUB1 leads to decreased modification of p53 with NEDD8 and stimulation of p53 ubiquitination. The biological outcome is the cytoplasmic localization and inhibition of the transcriptional activity of p53. Although the effects of NUB1 on p53 depend on NEDDylation and the murine double minute 2 (Mdm2) E3-ligase, the cooperation of NEDD8 with ubiquitin is required. The data identify a role for NEDD8 in controlling p53 localization and suggest that NEDD8 can control protein function through its non-covalent recognition by interacting proteins.


GlcNAcylation plays an essential role in breast cancer metastasis.
GlcNAcylation, a dynamic posttranslational modification, is involved in a wide range of biological processes and some human diseases. Although there is emerging evidence that some tumor-associated proteins are modified by GlcNAcylation, the role of GlcNAcylation in tumor progression remains unclear. Here, we show that GlcNAcylation enhances the migration/invasion of breast cancer cells in vitro and lung metastasis in vivo. The decrease of cell surface E-cadherin is the molecular mechanism underlying GlcNAcylation-induced breast cancer metastasis. p120 and beta-catenin, but not E-cadherin, are GlcNAcylated; the GlcNAcylation of p120 and beta-catenin might play roles in the decrease of cell surface E-cadherin. Moreover, immunohistochemistry analysis indicated that the global GlcNAcylation level in breast tumor tissues is elevated significantly as compared with that in the corresponding adjacent tissues; further, GlcNAcylation was significantly enhanced in metastatic lymph nodes compared with their corresponding primary tumor tissues. This is the first report to clearly elucidate the roles and mechanisms whereby GlcNAcylation influences the malignant properties of breast cancer cells. These results also suggest that GlcNAcylation might be a potential target for the diagnosis and therapy of breast cancer.


Correlation between MRI and clinico-pathological manifestations in Lewis rats protected from experimental allergic encephalomyelitis by acylated synthetic peptide of myelin basic protein.
Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system which constitutes an accepted animal model for multiple sclerosis (MS). The disease can take an acute or chronic form depending on the injection route, animal strain and nature of the disease-inducing antigen administered. The neuroinflammation associated with the acute form can be detected with T2-weighted, T1-weighted and diffusion MRI, and blood-brain barrier changes can be investigated with Gd-DTPA-enhanced T1-weighted imaging, similar to that of MS patients. A synthetic peptide of myelin basic protein (MBP) encephalitogenic for the Lewis rat (MBP 68-86) was acylated by the attachment of a palmitoyl residue (PAL68-86), and was shown to confer almost complete protection against EAE, when administered to rats before and after an encephalitogenic challenge. In this study, treatment of Lewis rats with PAL68-86 prevented the appearance of clinical signs (p < 0.0001) after challenge with the native peptide (p68-86) in complete Freund's adjuvant (CFA), and reduced considerably the MRI and histopathological signs of the disease (p < 0.0001). Measurement of the gadolinium leakage due to neuroinflammation revealed a significant decrease in permeability from 4.09 +/- 2.1 to 2.95 +/- 1.79% pixels > mean + 2 SD (p = 0.011). Therefore, quantitative MRI measurements correlate very well with the reduced cellular infiltration in the CNS and the absence of clinical signs in the EAE-protected animal.


The acylation of megakaryocyte proteins: glycoprotein IX is primarily myristoylated while glycoprotein Ib is palmitoylated.
The acylation of megakaryocyte proteins was studied with special emphasis on the myristoylation and palmitoylation of the glycoprotein (GP) Ib complex. Guinea pig megakaryocytes were purified and separated into subpopulations at different phases of maturation. Cells were incubated with [3H]myristate, [3H]palmitate, or [3H]acetate to study endogenous protein acylation. Cycloheximide was used to distinguish between cotranslational and posttranslational acylation and hydroxylamine to distinguish between thioester and amide linkages. After incubations, delipidated proteins or GPIb complex subunits, immunoprecipitated with PG-1, AN-51 or FMC-25 monoclonal antibody, were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and assessed by fluorography. Radiolabeled fatty acids bound to GPIX and GPIb were also analyzed by high pressure liquid chromatography (HPLC) and scintillation spectrometry. With [3H]myristic acid and [3H]acetate, GPIX was found to be a major myristoylated protein in megakaryocytes and CHRF-288 cells. Myristic acid was linked to GPIX by an amide bond, and this process occurred cotranslationally. With [3H]acetate, GPIb was primarily palmitoylated, but with [3H]myristate, GPIb was acylated with about equal mounts of myristic acid and palmitic acids. Both fatty acids were linked to GPIb by thioester bonds, and acylation was posttranslational. The myristoylation of GPIX while the palmitoylation of GPIb occurred throughout megakaryocyte maturation. Myristoylation and palmitoylation may have different functions relevant to the assembly of the GPIb complex in megakaryocytes.


cDNA cloning of component A of Rab geranylgeranyl transferase and demonstration of its role as a Rab escort protein.
cDNA cloning of component A of rat Rab geranylgeranyl transferase confirms identity of the protein with the human choroideremia gene product and its resemblance to Rab3A guanine nucleotide dissociation inhibitor (GDI), which binds prenylated Rabs. In biochemical assays we demonstrate that component A binds unprenylated Rab1A, presents it to the catalytic component B, and remains bound to it after the geranylgeranyl transfer reaction. In the absence of detergents, the reaction terminates when all of component A is occupied with prenylated Rab. Detergents allow multiple rounds of catalysis, apparently by dissociating the component A-Rab complex and thus allowing recycling of component A. Within the cell, component A may be regenerated by transferring its prenylated Rab to a protein acceptor, such as Rab3A GDI. In view of its function in escorting Rab proteins during and presumably after the prenyl transfer reaction, we propose to rename component A as Rab escort protein (REP). A genetic defect in REP underlies human choroideremia, a disease of retinal degeneration.


Drugs affecting prelamin A processing: effects on heterochromatin organization.
Increasing interest in drugs acting on prelamin A has derived from the finding of prelamin A involvement in severe laminopathies. Amelioration of the nuclear morphology by inhibitors of prelamin A farnesylation has been widely reported in progeroid laminopathies. We investigated the effects on chromatin organization of two drugs inhibiting prelamin A processing by an ultrastructural and biochemical approach. The farnesyltransferase inhibitor FTI-277 and the non-peptidomimetic drug N-acetyl-S-farnesyl-l-cysteine methylester (AFCMe) were administered to cultured control human fibroblasts for 6 or 18 h. FTI-277 interferes with protein farnesylation causing accumulation of non-farnesylated prelamin A, while AFCMe impairs the last cleavage of the lamin A precursor and is expected to accumulate farnesylated prelamin A. FTI-277 caused redistribution of heterochromatin domains at the nuclear interior, while AFCMe caused loss of heterochromatin domains, increase of nuclear size and nuclear lamina thickening. At the biochemical level, heterochromatin-associated proteins and LAP2 alpha were clustered at the nuclear interior following FTI-277 treatment, while they were unevenly distributed or absent in AFCMe-treated nuclei. The reported effects show that chromatin is an immediate target of FTI-277 and AFCMe and that dramatic remodeling of chromatin domains occurs following treatment with the drugs. These effects appear to depend, at least in part, on the accumulation of prelamin A forms, since impairment of prelamin A accumulation, here obtained by 5-azadeoxycytidine treatment, abolishes the chromatin effects. These results may be used to evaluate downstream effects of FTIs or other prelamin A inhibitors potentially useful for the therapy of laminopathies.


A novel N-terminal motif for palmitoylation of G-protein alpha subunits.
We have examined the post-translational processing of G alpha subunits expressed endogenously in rat PC12 and NG108-15 rat/mouse hybrid cells, and after transfection of cDNA expression constructs into COS cells. Thioester-linked palmitoylation of alpha o, alpha s, alpha q/alpha 11 and alpha 12 has been detected by metabolic labelling with [3H]palmitate and immunoprecipitation. Palmitoylation of alpha o occurs post-translationally in cells treated with protein-synthesis inhibitors, suggesting possible dynamic acylation. Palmitoylation of the C-terminal CAAX motif has been excluded. Site-directed mutagenesis of alpha o has been used to implicate the site of modification as a cysteine residue next to the N-terminal myristoylated glycine, in a novel protein-lipid modification motif Met-Gly-Cys. The non-palmitoylated alpha o mutant is still myristoylated but shows reduced membrane binding, suggesting that reversible palmitoylation may regulate G alpha localization and function.


Xenobiotic incorporation into pyruvate dehydrogenase complex can occur via the exogenous lipoylation pathway.
Lipoylated enzymes such as the E2 component of pyruvate dehydrogenase complex (PDC-E2) are targets for autoreactive immune responses in primary biliary cirrhosis, with lipoic acid itself forming a component of the dominant auto-epitopes. A candidate mechanism for the initiation of tolerance breakdown in this disease is immune recognition of neo-antigens formed by xenobiotic substitution of normal proteins. Importantly, sensitization with proteins artificially substituted with the lipoic acid analogue xenobiotic 6-bromohexanoic acid (6BH) can induce an immune response that cross-reacts with PDC-E2. This study investigated the potential of recombinant lipoylation enzymes lipoate activating enzyme and lipoyl-AMP(GMP):N-lysine lipoyl transferase to aberrantly incorporate xenobiotics into PDC-E2. It was found that these enzymes could incorporate lipoic acid analogues including octanoic and hexanoic acids and the xenobiotic 6BH into PDC-E2. The efficiency of incorporation of these analogues showed a variable dependence on activation by adenosine triphosphate (ATP) or guanosine triphosphate (GTP), with ATP favoring the incorporation of hexanoic acid and 6BH whereas GTP enhanced substitution by octanoic acid. Importantly, competition studies showed that the relative incorporation of both 6BH and lipoic acid could be regulated by the balance between ATP and GTP, with the formation of 6BH-substituted PDC-E2 predominating in an ATP-rich environment. Conclusion: Using a well-defined system in vitro we have shown that an important xenobiotic can be incorporated into PDC in place of lipoic acid by the exogenous lipoylation system; the relative levels of lipoic acid and xenobiotic incorporation may be determined by the balance between ATP and GTP. These observations suggest a clear mechanism for the generation of an auto-immunogenic neo-antigen of relevance for the pathogenesis of primary biliary cirrhosis.


Rate limitations in posttranslational processing by the mammary gland of transgenic animals.
Our studies in transgenic animal bioreactors sought to determine the rate limitations in posttranslational processing of recombinant human protein C (rhPC) made in mammary gland of mice and pigs. Human protein C (hPC) is a complex plasma protein containing nine gamma-carboxylated glutamic acid (gla) residues that bind calcium at about 1 to 3 mM. Gamma carboxylation is a vitamin K-dependent posttranslational modification. The effect of rhPC synthesis rate on the extent of gamma-carboxylation of glutamic acid was studied. We have perturbed the biosynthesis of rhPC by using two different transgenes to direct mammary gland-specific expression. Promoter elements of the murine whey acid protein (mWAP) gene were used to drive the expression of hPC-cDNA and hPC-genomic transgenes. Transgenic mice with hPC-cDNA and hPC-genomic sequences gave expression levels of 11 +/- 4 micrograms rhPC/ml of milk and 895 +/- 21 micrograms rhPC/ml of milk, respectively. Transgenic pigs with hPC-cDNA and hPC-genomic sequences gave expression levels of 100 to 500 micrograms rhPC/ml of milk and 800 to 2000 micrograms rhPC/ml of milk, respectively. A monoclonal antibody (7D7B10-mAb) that binds an epitope in the gla domain of hPC in the absence of calcium was used to study the conformational behavior of immunopurified rhPC. Immunopurified rhPC from lower expressing mice and pigs gave a calcium-dependent binding inhibition by 7D7B10-mAb similar to that of hPC. Immunopurified rhPC from higher expressing mice and pigs gave a less calcium-dependent response. This study suggests that a rate limitation in gamma-carboxylation by the mammary gland occurs at expression levels about > 20 micrograms/ml in mice and > 500 micrograms/ml in pigs.


Tyrosine sulfation: an increasingly recognised post-translational  modification of secreted proteins.
The post-translational sulfation of tyrosine residues occurs in numerous secreted and integral membrane proteins and, in many cases, plays a crucial role in controlling the interactions of these proteins with physiological binding partners as well as invading pathogens. Recent advances in our understanding of protein tyrosine sulfation have come about owing to the cloning of two human tyrosylprotein sulfotransferases (TPST-1 and TPST-2), the development of novel analytical and synthetic methodologies and detailed studies of proteins and peptides containing sulfotyrosine residues. In this article, we describe the TPST enzymes, review the major techniques available for studying the presence, location and function of tyrosine sulfation in proteins and discuss the biological functions and biochemical interactions of several proteins (or protein families) in which tyrosine sulfation influences the protein function. In particular, we describe the detailed evidence supporting the importance of tyrosine sulfation in the cellular adhesion function of P-selectin glycoprotein ligand-1, the leukocyte trafficking and pathogen invasion functions of chemokine receptors and the ligand binding and activation of other G-protein-coupled receptors by complement proteins, phospholipdis and glycoprotein hormones.


Iontophoresis of lysophosphatidic acid into rabbit cornea induces HSV-1 reactivation: evidence that neuronal signaling changes after infection.
PURPOSE: Lysophosphatidic acid induces neurite retraction; it is also present in tears and aqueous humor. We determined whether lysophosphatidic acid induces HSV-1 reactivation in latently infected rabbits and whether the nerve growth associated protein GAP-43 undergoes posttranslational modification during the course of HSV-1 infection. METHODS: Rabbits were infected with HSV-1 and acute infection was documented by slit lamp examination. Corneas of latently infected rabbits were treated with lysophosphatidic acid or lysophosphatidylserine (structurally similar but lacking biological potency). For application to the cornea, these compounds were impregnated into collagen shields, applied as topical drops, or iontophoresed. In another experiment, corneas of latently infected rabbits were either untreated or treated iontophoretically with lysophosphatidic acid, lysophosphatidylserine, or saline. Ocular swabs detected shedding of infectious virus. Western blot and immunoprecipitation identified GAP-43 in corneal extracts and densitometry of silver-stained isoelectric focusing gels measured changes in GAP-43 isoform abundance. RESULTS: Iontophoresis of lysophosphatidic acid induced HSV-1 shedding more frequently than lysophosphatidylserine or saline. Viral shedding induced by collagen shield and topical drop administration was low and not significantly different for lysophosphatidic acid and lysophosphatidylserine. Five discrete GAP-43 isoforms predominated in the IEF gels. Most abundant were the pI 4.7 band in uninfected cornea and the pI 5.05 band in latently-infected cornea. Compared to latently-infected cornea, there was no significant change in isoform abundance 1 h after lysophosphatidic acid iontophoresis, but 24 and 72 h later, the pI 5. 05 band was diminished. CONCLUSIONS: Lysophosphatidic acid can induce HSV-1 reactivation and changes in GAP-43 pI suggest that posttranslational modifications, possibly related to phosphorylation and ADP-ribosylation, are occurring during HSV-1 latency and after LPA is iontophoretically applied to the cornea. How lysophosphatidic acid-induced signaling, HSV-1 reactivation, and GAP-43 pI are related remains to be determined.


Subcellular localization directs signaling specificity of the Cryptococcus neoformans Ras1 protein.
In the human fungal pathogen Cryptococcus neoformans, Ras signaling mediates sexual differentiation, morphogenesis, and pathogenesis. By studying Ras prenylation and palmitoylation in this organism, we have found that the subcellular localization of this protein dictates its downstream signaling specificity. Inhibiting C. neoformans Ras1 prenylation results in the defective general membrane targeting of this protein and the loss of all Ras function. In contrast, palmitoylation mediates localization of Ras1 to the plasma membrane and is required for normal morphogenesis and survival at high temperatures. However, palmitoylation and plasma membrane localization are not required for Ras-dependent sexual differentiation. Likely as a result of its effect on thermotolerance, Ras1 palmitoylation is also required for the pathogenesis of C. neoformans. These data support an emerging paradigm of compartmentalized Ras signaling. However, our studies also demonstrate fundamental differences between the Ras pathways in different organisms that emphasize the functional flexibility of conserved signaling cascades.


Thiopalmitoylation of myelin proteolipid protein epitopes enhances immunogenicity and encephalitogenicity.
Proteolipid protein (PLP) is the most abundant protein of CNS myelin, and is posttranslationally acylated by covalent attachment of long chain fatty acids to cysteine residues via a thioester linkage. Two of the acylation sites are within epitopes of PLP that are encephalitogenic in SJL/J mice (PLP(104-117) and PLP(139-151)) and against which increased immune responses have been detected in some multiple sclerosis patients. It is known that attachment of certain types of lipid side chains to peptides can result in their enhanced immunogenicity. The aim of this study was to determine whether thioacylated PLP peptides, as occur in the native protein, are more immunogenic than their nonacylated counterparts, and whether thioacylation influences the development of autoreactivity and experimental autoimmune encephalomyelitis. The results show that in comparison with nonacylated peptides, thioacylated PLP lipopeptides can induce greater T cell and Ab responses to both the acylated and nonacylated peptides. They also enhanced the development and chronicity of experimental autoimmune encephalomyelitis. Synthetic peptides in which the fatty acid was attached via an amide linkage at the N terminus were not encephalitogenic, and they induced greater proportions of CD8+ cells in initial in vitro stimulation. Therefore, the lability and the site of the linkage between the peptide and fatty acid may be important for induction of encephalitogenic CD4+ T cells. These results suggest that immune responses induced by endogenous thioacylated lipopeptides may contribute to the immunopathogenesis of chronic experimental demyelinating diseases and multiple sclerosis.


Dynamic compartmentalization of base excision repair proteins in response to nuclear and mitochondrial oxidative stress.
DNAs harbored in both nuclei and mitochondria of eukaryotic cells are subject to continuous oxidative damage resulting from normal metabolic activities or environmental insults. Oxidative DNA damage is primarily reversed by the base excision repair (BER) pathway, initiated by N-glycosylase apurinic/apyrimidinic (AP) lyase proteins. To execute an appropriate repair response, BER components must be distributed to accommodate levels of genotoxic stress that may vary considerably between nuclei and mitochondria, depending on the growth state and stress environment of the cell. Numerous examples exist where cells respond to signals, resulting in relocalization of proteins involved in key biological transactions. To address whether such dynamic localization contributes to efficient organelle-specific DNA repair, we determined the intracellular localization of the Saccharomyces cerevisiae N-glycosylase/AP lyases, Ntg1 and Ntg2, in response to nuclear and mitochondrial oxidative stress. Fluorescence microscopy revealed that Ntg1 is differentially localized to nuclei and mitochondria, likely in response to the oxidative DNA damage status of the organelle. Sumoylation is associated with targeting of Ntg1 to nuclei containing oxidative DNA damage. These studies demonstrate that trafficking of DNA repair proteins to organelles containing high levels of oxidative DNA damage may be a central point for regulating BER in response to oxidative stress.


HSDJ, a human homolog of DnaJ, is farnesylated and is involved in protein import into mitochondria.
The role of HSDJ, a human homolog of bacterial DnaJ and yeast YDJ1p/MAS5, in mitochondrial protein import was examined. Recombinant HSDJ was purified and an antibody was prepared. HSDJ mRNA was heat-induced in cultured cells. In pulse-labeling and chase experiments using COS-7 cells, the endogenous HSDJ homolog was prenylated. Transiently expressed HSDJ was also prenylated, whereas its mutant C394S in which cysteine of the "CaaX box" was mutated to serine, was not. HSDJ, but not C394S, synthesized in rabbit reticulocyte lysate was farnesylated. The HSDJ antibody inhibited import of ornithine transcarbamylase precursor (pOTC) into isolated mitochondria when added prior to pOTC synthesis, but not when added prior to import assay. In transient expression of pOTC in COS-7 cells, pOTC was synthesized and processed to the mature form with an apparent half-life of 2-3 min. Coexpression of HSDJ or C394S resulted in slight retardation of the pOTC processing. These results indicate that HSDJ is involved in an early step(s) of protein import into mitochondria.


Elucidation of XA21-mediated innate immunity.
In the early 1970s, the Xa21 gene from the wild rice species Oryza longistaminata drew attention of rice breeders because of its broad-spectrum resistance to diverse strains of a serious bacterial disease of rice in Asia and Africa, called 'bacterial blight disease', caused by the Gram-negative bacterium, Xanthomonas oryzae pv. oryzae (Xoo). In 1995, we isolated the gene controlling this resistance and in 2009 demonstrated that XA21 recognizes a highly conserved peptide, called 'Ax21' (activator of XA21-mediated immunity). Tyrosine sulfation of Ax21 is required for recognition by rice XA21. A decade of genetic, molecular and biochemical studies have uncovered key components of the XA21-mediated signalling cascade. Ax21 recognition by XA21 at the cell surface induces phosphorylation-mediated events, which are predicted to alter subcellular localization and/or DNA-binding activity of a WRKY family of transcription factors. Because XA21 is representative of the large number of predicted pattern recognition receptors (PRRs) in rice (n = 328), Arabidopsis (n = 35) and other plant species, further characterization of XA21-mediated signalling pathways will contribute to elucidation of these important defence responses.


Purification and postsynthetic modifications of Friend erythroleukemic cell high mobility group protein HMG-I.
We have previously detected and purified a Friend erythroleukemic mouse cell nonhistone chromatin protein having extraction and acid-solubility properties like the low molecular weight "high mobility group" (HMG) nuclear proteins. We show here that the electrophoretic properties and the amino acid composition of this mouse cell "HMG-like" protein is comparable to those of the HMG-I proteins isolated from human HeLa S3 cells, African green monkey cells, Ehrlich ascites mouse cells, and rat fibroblast cells. Therefore, we have also designated the Friend erythroleukemic mouse cell protein as HMG-I. In common with the other HMG proteins the Friend cell HMG-I protein can undergo a variety of post-translational biochemical modifications including acetylation, ADP-ribosylation, glycosylation, and phosphorylation. Surprisingly, in the course of these studies we found that in vivo radiolabeling experiments revealed that only two minor HMG-14 subspecies (and/or possibly a minor HMG-I subspecies) are phosphorylated whereas HMG-1, -2, -17, and the major HMG-14 are not heavily phosphorylated.


The effects of processing inhibitors of N-linked oligosaccharides on the intracellular migration of glycoprotein E2 of mouse hepatitis virus and the maturation of coronavirus particles.
We have studied the effects of tunicamycin and inhibitors of the processing of N-linked glycans including N-methyl-1-deoxynojirimycin, castanospermine, mannodeoxynojirimycin, and swainsonine on the transport of glycoprotein E2 and the intracellular maturation of the coronavirus mouse hepatitis virus A59. Indirect immunofluorescence staining with monoclonal antibodies revealed that glycoprotein E2 exhibits different antigenic properties depending on the presence and on the structure of the N-linked oligosaccharides and that efficient transport of glycoprotein E2 to the plasma membrane requires the removal of glucose residues. In the presence of tunicamycin in the nonglycosylated E2 apoprotein was synthesized in normal amounts and readily acylated throughout the infectious cycle. This E2-species could not be detected on the surface of mouse hepatitis virus A59-infected cells with indirect immunofluorescence staining or lactoperoxidase labeling. N-Methyl-1-deoxynojirimycin and castanospermine, both of which selectively inhibited the processing glucosidases, caused a drop in virion formation by two log steps and a drastic delay in the surface expression of glycoprotein E2. The E2 species synthesized under such conditions was acylated but accumulated intracellularly in a compartment distinct from the Golgi. Concomitantly, synthesis of the matrix glycoprotein E1 of mouse hepatitis virus A59 was drastically impaired. Mannodeoxynojirimycin and swainsonine, which block later stages of the processing pathway, had less or no effect on the transport of glycoprotein E2 and the formation of virus particles.


Isoprenylation of large hepatitis delta antigen is necessary but not sufficient for hepatitis delta virus assembly.
Hepatitis delta virus (HDV) encodes two proteins, the small hepatitis delta antigen (SHDAg) and large hepatitis delta antigen (LHDAg). Both proteins are identical except for the presence of additional 19 amino acids at the C terminus of LHDAg. While SHDAg is required for HDV RNA replication, LHDAg inhibits replication and is required together with hepatitis B surface antigen for the assembly of HDV. The C-terminal last 4 amino acids of LHDAg (Cys-Arg-Pro-Gln) is an isoprenylation motif. It has previously been shown that the mutation of the Cys inhibited the assembly of HDV. In order to discern whether this effect is due to change of amino acid residue or abolition of isoprenylation, we constructed several LHDAg mutants of the terminal three amino acid residues and tested their abilities to be packaged with HBsAg by cotransfection experiments. We also made GST-fusion proteins of these mutants and tested their abilities to be isoprenylated in rabbit reticulocyte lysate system. We found that some, but not all, of the substitutions of the amino acid residues other than the Cys also inhibited isoprenylation and that the status of isoprenylation of these mutant proteins correlated well with their abilities to be packaged with HBsAg into virions. This result indicates that isoprenylation, rather than the primary amino acid sequence, is required for LHDAg packaging. Furthermore, we found that the attachment of an isoprenylation motif to SHDAg did not enable it to be packaged with HBsAg and that the deletions of any 5 amino acids in the last 15 amino acids (amino acids 196 to 210) unique to the LHDAg abolished the packaging ability. In contrast, the deletion of 33 amino acids (amino acids 163 to 195) upstream of the last C-terminal 19 amino acids of LHDAg did not interfere with its packaging ability. Therefore, we conclude that the 15 amino acids upstream of the isoprenylation site of LHDAg are also essential for HDV assembly, and a large portion of the alleged C-terminal Pro/Gly-rich region (amino acids 146 to 195) is not required for the assembly process.


SUMOylation attenuates the aggregation propensity and cellular toxicity of the polyglutamine expanded ataxin-7.
Post-translational modification by SUMO (small ubiquitin-like modifier) was proposed to modulate the pathogenesis of several neurodegenerative diseases. Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disorder, whose pathology is caused by an expansion of a polyglutamine stretch in the protein ataxin-7 (ATXN7). Here, we identified ATXN7 as new target for SUMOylation in vitro and in vivo. The major SUMO acceptor site was mapped to lysine 257, which is part of an evolutionarily conserved consensus SUMOylation motif. SUMOylation did not influence the subcellular localization of ATXN7 nor its interaction with components of the TFTC/STAGA complex. Expansion of the polyglutamine stretch did not impair the SUMOylation of ATXN7. Furthermore, SUMO1 and SUMO2 colocalized with ATXN7 in a subset of neuronal intranuclear inclusions in the brain of SCA7 patients and SCA7 knock-in mice. In a COS-7 cellular model of SCA7, in addition to diffuse nucleoplasmic staining we identified two populations of nuclear inclusions: homogenous or non-homogenous. Non-homogenous inclusions showed significantly reduced colocalization with SUMO1 and SUMO2, but were highly enriched in Hsp70, 19S proteasome and ubiquitin. Interestingly, they were characterized by increased staining with the apoptotic marker caspase-3 and by disruption of PML nuclear bodies. Importantly, preventing the SUMOylation of expanded ATXN7 by mutating the SUMO site increased both the amount of SDS-insoluble aggregates and of caspase-3 positive non-homogenous inclusions, which act toxic to the cells. Our results demonstrate an influence of SUMOylation on the multistep aggregation process of ATXN7 and implicate a role for ATXN7 SUMOylation in SCA7 pathogenesis.


The human receptor for T-cell growth factor. Evidence for variable post-translational processing, phosphorylation, sulfation, and the ability of precursor forms of the receptor to bind T-cell growth factor.
The T-cell growth factor (TCGF) receptor on phytohemagglutinin-activated normal peripheral blood T-cells is characterized as a glycoprotein with an apparent Mr = 55,000 that contains N-linked and O-linked carbohydrate with only approximately 33,000 daltons of peptide structure (p33) as evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There are two N-linked glycosylated intermediate precursor forms (apparent Mr = 35,000 (p35) and 37,000 (p37]. This receptor differs from the TCGF receptor on HUT-102B2 cells (apparent Mr = 50,000) because of differences in post-translational processing. Experiments with the carboxylic ionophore monensin demonstrate blockade of the transition of the p35 and p37 receptor precursor forms to the mature receptor, presumably secondary to inhibition of Golgi-associated receptor processing. We identify the primary translation product of TCGF receptor mRNA as intermediate in size between the p33 and the p35/p37 forms. We further demonstrate that the p33, p35, and p37 precursor forms, but not the primary translation product, are all capable of binding TCGF. Thus, the removal of the signal peptide and/or conformational changes of the primary translation product are necessary for ligand binding; however, the extensive post-translational modifications are not. Lastly, we demonstrate that at least some TCGF receptors are phosphorylated and sulfated, and that TCGF receptors on phytohemagglutinin-activated normal T-cells are more heavily sulfated than those on HUT-102B2 cells.


In vitro and in vivo functional characterization of bovine vitamin K-dependent gamma-carboxylase expressed in Chinese hamster ovary cells.
Coagulation factor IX is a serine protease for which high-level expression of biologically active protein in heterologous cells is limited due to inefficient proteolytic removal of the propeptide as well as vitamin K-dependent carboxylation of multiple amino-terminal glutamic acid residues. We have overexpressed the vitamin K-dependent gamma-carboxylase cDNA and monitored its ability to improve factor IX processing in Chinese hamster ovary (CHO) cells. From amino acid sequence analysis of bovine liver vitamin K-dependent gamma-carboxylase, degenerate oligonucleotides were used to isolate a 3.5-kbp bovine cDNA that encoded a 758-residue open reading frame. Expression of the cDNA in COS-1 and CHO cells yielded 17- and 16-fold increases in the in vitro gamma-carboxylase activity of microsomal preparations, respectively. Anti-serum raised against a predicted peptide sequence reacted with a 94-kDa polypeptide in the partially purified bovine liver preparation as well as in stably transfected CHO cells. The amount of antibody reactivity correlated with the increased ability to carboxylate a peptide substrate in vitro. These results strongly support the conclusion that the cDNA encodes the vitamin K-dependent gamma-carboxylase. Transient transfection of the gamma-carboxylase expression vector into factor IX-expressing CHO cells did not improve the specific procoagulant activity of secreted factor IX. In contrast, transfection of an expression vector encoding the propeptide processing enzyme PACE (paired basic amino acid cleaving enzyme) did improve the specific activity of secreted factor IX by 3-fold. These results demonstrate that the ability of CHO cells to modify glutamic acid residues to gamma-carboxyglutamic acid in secreted factor IX is not limited by the expression of the vitamin K-dependent gamma-carboxylase alone.


A comparative kinetic study on the singlet molecular oxygen-mediated photoxidation of alpha- and beta-chymotrypsins.
Kinetic aspects of the sensitized photooxidation of alpha- and beta-chymotrypsins have been studied at pH 6 and 8. The sensitization, employing classical O2(1Deltag)-photogenerators, such as xanthene dyes, is a kinetically intricate process because of the presence of ground state dye-protein associations and to the simultaneous participation of superoxide ion and singlet molecular oxygen [O2(1Deltag)]. Both proteins, that possess the same distribution pattern of photooxidizable amino acids, suffer a pure O2(1Deltag)-mediated photodynamic attack, using the carbonylic sensitizer Perinaphthenone. Overall and reactive rate constants for the O2(1Deltag)-quenching (in the order of 108 and 107/M/s, respectively), and rates of oxygen consumption determined by time-resolved, spectroscopic and polarographic methods indicate that alpha- and beta-chymotrypsins are less photooxidizable at pH 6, as a result of an enhancement of the O2(1Deltag)-physical quenching component. In general terms, beta-chymotrypsin exhibits the greater overall proclivity to interact with O2(1Deltag), whereas structural factors, possibly evidenced by a higher exposure of the reactive tryptophan residues, impart an increased photooxidation degree to the proteins at pH 8, specially to the alpha-chymotrypsin.