Generation of secretable and nonsecretable interleukin 15 isoforms through alternate usage of signal peptides

Two isoforms of human interleukin 15 (IL-15) exist. One isoform has a shorter putative signal peptide (21 amino acids) and its transcript shows a tissue distribution pattern that is distinct from that of the alternative IL-15 isoform with a 48-aa signal peptide. The 21-aa signal isoform is preferentially expressed in tissues such as testis and thymus. Experiments using different combinations of signal peptides and mature proteins (IL-2, IL-15, and green fluorescent protein) showed that the short signal peptide regulates the fate of the mature protein by controlling the intracellular trafficking to nonendoplasmic reticulum sites, whereas the long signal peptide both regulates the rate of protein translation and functions as a secretory signal peptide. As a consequence, the IL-15 associated with the short signal peptide is not secreted, but rather is stored intracellularly, appearing in the nucleus and cytoplasmic components. Such production of an intracellular lymphokine is not typical of other soluble interleukin systems, suggesting a biological function for IL-15 as an intracellular molecule.

RacF1, a Novel Member of the Rho Protein Family in Dictyostelium discoideum, Associates Transiently with Cell Contact Areas, Macropinosomes, and Phagosomes

Using a PCR approach we have isolated racF1, a novel member of the Rho family in Dictyostelium. The racF1 gene encodes a protein of 193 amino acids and is constitutively expressed throughout the Dictyostelium life cycle. Highest identity (94%) was found to a RacF2 isoform, to Dictyostelium Rac1A, Rac1B, and Rac1C (70%), and to Rac proteins of animal species (64–69%). To investigate the role of RacF1 in cytoskeleton-dependent processes, we have fused it at its amino-terminus with green fluorescent protein (GFP) and studied the dynamics of subcellular redistribution using a confocal laser scanning microscope and a double-view microscope system. GFP–RacF1 was homogeneously distributed in the cytosol and accumulated at the plasma membrane, especially at regions of transient intercellular contacts. GFP–RacF1 also localized transiently to macropinosomes and phagocytic cups and was gradually released within <1 min after formation of the endocytic vesicle or the phagosome, respectively. On stimulation with cAMP, no enrichment of GFP–RacF1 was observed in leading fronts, from which it was found to be initially excluded. Cell lines were obtained using homologous recombination that expressed a truncated racF1 gene lacking sequences encoding the carboxyl-terminal region responsible for membrane targeting. These cells displayed normal phagocytosis, endocytosis, and exocytosis rates. Our results suggest that RacF1 associates with dynamic structures that are formed during pinocytosis and phagocytosis. Although RacF1 appears not to be essential, it might act in concert and/or share functions with other members of the Rho family in the regulation of a subset of cytoskeletal rearrangements that are required for these processes.

Protective DNA vaccination against organ-specific autoimmunity is highly specific and discriminates between single amino acid substitutions in the peptide autoantigen

DNA vaccines that encode encephalitogenic sequences in tandem can protect from subsequent experimental autoimmune encephalomyelitis induced with the corresponding peptide. The mechanism for this protection and, in particular, if it is specific for the amino acid sequence encoding the vaccine are not known. We show here that a single amino acid exchange in position 79 from serine (nonself) to threonine (self) in myelin basic protein peptide MBP68–85, which is a major encephalitogenic determinant for Lewis rats, dramatically alters the protection. Moreover, vaccines encoding the encephalitogenic sequence MBP68–85 do not protect against the second encephalitogenic sequence MBP89–101 in Lewis rats and vice versa. Thus, protective immunity conferred by DNA vaccination exquisitely discriminates between peptide target autoantigens. No bystander suppression was observed. The exact underlying mechanisms remain elusive because no simple correlation between impact on ex vivo responses and protection against disease were noted.

CXR, a chicken xenobiotic-sensing orphan nuclear receptor, is related to both mammalian pregnane X receptor (PXR) and constitutive androstane receptor (CAR)

Nuclear receptors constitute a large family of ligand-modulated transcription factors that mediate cellular responses to small lipophilic molecules, including steroids, retinoids, fatty acids, and exogenous ligands. Orphan nuclear receptors with no known endogenous ligands have been discovered to regulate drug-mediated induction of cytochromes P450 (CYP), the major drug-metabolizing enzymes. Here, we report the cloning of an orphan nuclear receptor from chicken, termed chicken xenobiotic receptor (CXR), that is closely related to two mammalian xenobiotic-activated receptors, the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR). Expression of CXR is restricted to tissues where drug induction of CYPs predominantly occurs, namely liver, kidney, small intestine, and colon. Furthermore, CXR binds to a previously identified phenobarbital-responsive enhancer unit (PBRU) in the 5′-flanking region of the chicken CYP2H1 gene. A variety of drugs, steroids, and chemicals activate CXR in CV-1 monkey cell transactivation assays. The same agents induce PBRU-dependent reporter gene expression and CYP2H1 transcription in a chicken hepatoma cell line. These results provide convincing evidence for a major role of CXR in the regulation of CYP2H1 and add a member to the family of xenobiotic-activated orphan nuclear receptors.

Structural code for DNA recognition revealed in crystal structures of papillomavirus E2-DNA targets

Transcriptional regulation in papillomaviruses depends on sequence-specific binding of the regulatory protein E2 to several sites in the viral genome. Crystal structures of bovine papillomavirus E2 DNA targets reveal a conformational variant of B-DNA characterized by a roll-induced writhe and helical repeat of 10.5 bp per turn. A comparison between the free and the protein-bound DNA demonstrates that the intrinsic structure of the DNA regions contacted directly by the protein and the deformability of the DNA region that is not contacted by the protein are critical for sequence-specific protein/DNA recognition and hence for gene-regulatory signals in the viral system. We show that the selection of dinucleotide or longer segments with appropriate conformational characteristics, when positioned at correct intervals along the DNA helix, can constitute a structural code for DNA recognition by regulatory proteins. This structural code facilitates the formation of a complementary protein–DNA interface that can be further specified by hydrogen bonds and nonpolar interactions between the protein amino acids and the DNA bases.

Uncoupling proteins 2 and 3 are highly active H+ transporters and highly nucleotide sensitive when activated by coenzyme Q (ubiquinone)

Based on the discovery of coenzyme Q (CoQ) as an obligatory cofactor for H+ transport by uncoupling protein 1 (UCP1) [Echtay, K. S., Winkler, E. & Klingenberg, M. (2000) Nature (London) 408, 609–613] we show here that UCP2 and UCP3 are also highly active H+ transporters and require CoQ and fatty acid for H+ transport, which is inhibited by low concentrations of nucleotides. CoQ is proposed to facilitate injection of H+ from fatty acid into UCP. Human UCP2 and 3 expressed in Escherichia coli inclusion bodies are solubilized, and by exchange of sarcosyl against digitonin, nucleotide binding as measured with 2′-O-[5-(dimethylamino)naphthalene-1-sulfonyl]-GTP can be restored. After reconstitution into vesicles, Cl− but no H+ are transported. The addition of CoQ initiates H+ transport in conjunction with fatty acids. This increase is fully sensitive to nucleotides. The rates are as high as with reconstituted UCP1 from mitochondria. Maximum activity is at a molar ratio of 1:300 of CoQ:phospholipid. In UCP2 as in UCP1, ATP is a stronger inhibitor than ADP, but in UCP3 ADP inhibits more strongly than ATP. Thus UCP2 and UCP3 are regulated differently by nucleotides, in line with their different physiological contexts. These results confirm the regulation of UCP2 and UCP3 by the same factors CoQ, fatty acids, and nucleotides as UCP1. They supersede reports that UCP2 and UCP3 may not be H+ transporters.

An essential role for nuclear receptors SXR/PXR in detoxification of cholestatic bile acids

Hepatic hydroxylation is an essential step in the metabolism and excretion of bile acids and is necessary to avoid pathologic conditions such as cholestasis and liver damage. In this report, we demonstrate that the human xenobiotic receptor SXR (steroid and xenobiotic receptor) and its rodent homolog PXR (pregnane X receptor) serve as functional bile acid receptors in both cultured cells and animals. In particular, the secondary bile acid derivative lithocholic acid (LCA) is highly hepatotoxic and, as we show here, a metabolic substrate for CYP3A hydroxylation. By using combinations of knockout and transgenic animals, we show that activation of SXR/PXR is necessary and sufficient to both induce CYP3A enzymes and confer resistance to toxicity by LCA, as well as other xenotoxicants such as tribromoethanol and zoxazolamine. Therefore, we establish SXR and PXR as bile acid receptors and a role for the xenobiotic response in the detoxification of bile acids.

Purification and cDNA Cloning of Isochorismate Synthase from Elicited Cell Cultures of Catharanthus roseus

Isochorismate is an important metabolite formed at the end of the shikimate pathway, which is involved in the synthesis of both primary and secondary metabolites. It is synthesized from chorismate in a reaction catalyzed by the enzyme isochorismate synthase (ICS; EC 5.4.99.6). We have purified ICS to homogeneity from elicited Catharanthus roseus cell cultures. Two isoforms with an apparent molecular mass of 64 kD were purified and characterized. The Km values for chorismate were 558 and 319 μm for isoforms I and II, respectively. The isoforms were not inhibited by aromatic amino acids and required Mg2+ for enzyme activity. Polymerase chain reaction on a cDNA library from elicited C. roseus cells with a degenerated primer based on the sequence of an internal peptide from isoform II resulted in an amplification product that was used to screen the cDNA library. This led to the first isolation, to our knowledge, of a plant ICS cDNA. The cDNA encodes a protein of 64 kD with an N-terminal chloroplast-targeting signal. The deduced amino acid sequence shares homology with bacterial ICS and also with anthranilate synthases from plants. Southern analysis indicates the existence of only one ICS gene in C. roseus.

Depletion of Acyl-Coenzyme A-Binding Protein Affects Sphingolipid Synthesis and Causes Vesicle Accumulation and Membrane Defects in Saccharomyces cerevisiae

Deletion of the yeast gene ACB1 encoding Acb1p, the yeast homologue of the acyl-CoA-binding protein (ACBP), resulted in a slower growing phenotype that adapted into a faster growing phenotype with a frequency >1:105. A conditional knockout strain (Y700pGAL1-ACB1) with the ACB1 gene under control of the GAL1 promoter exhibited an altered acyl-CoA profile with a threefold increase in the relative content of C18:0-CoA, without affecting total acyl-CoA level as previously reported for an adapted acb1Δ strain. Depletion of Acb1p did not affect the general phospholipid pattern, the rate of phospholipid synthesis, or the turnover of individual phospholipid classes, indicating that Acb1p is not required for general glycerolipid synthesis. In contrast, cells depleted for Acb1p showed a dramatically reduced content of C26:0 in total fatty acids and the sphingolipid synthesis was reduced by 50–70%. The reduced incorporation of [3H]myo-inositol into sphingolipids was due to a reduced incorporation into inositol-phosphoceramide and mannose-inositol-phosphoceramide only, a pattern that is characteristic for cells with aberrant endoplasmic reticulum to Golgi transport. The plasma membrane of the Acb1p-depleted strain contained increased levels of inositol-phosphoceramide and mannose-inositol-phosphoceramide and lysophospholipids. Acb1p-depleted cells accumulated 50- to 60-nm vesicles and autophagocytotic like bodies and showed strongly perturbed plasma membrane structures. The present results strongly suggest that Acb1p plays an important role in fatty acid elongation and membrane assembly and organization.

Unsaturated fatty acids inhibit transcription of the sterol regulatory element-binding protein-1c (SREBP-1c) gene by antagonizing ligand-dependent activation of the LXR

Sterol regulatory element-binding protein-1c (SREBP-1c) enhances transcription of genes encoding enzymes of unsaturated fatty acid biosynthesis in liver. SREBP-1c mRNA is known to increase when cells are treated with agonists of liver X receptor (LXR), a nuclear hormone receptor, and to decrease when cells are treated with unsaturated fatty acids, the end products of SREBP-1c action. Here we show that unsaturated fatty acids lower SREBP-1c mRNA levels in part by antagonizing the actions of LXR. In cultured rat hepatoma cells, arachidonic acid and other fatty acids competitively inhibited activation of the endogenous SREBP-1c gene by an LXR ligand. Arachidonate also blocked the activation of a synthetic LXR-dependent promoter in transfected human embryonic kidney-293 cells. In vitro, arachidonate and other unsaturated fatty acids competitively blocked activation of LXR, as reflected by a fluorescence polarization assay that measures ligand-dependent binding of LXR to a peptide derived from a coactivator. These data offer a potential mechanism that partially explains the long-known ability of dietary unsaturated fatty acids to decrease the synthesis and secretion of fatty acids and triglycerides in livers of humans and other animals.

The spinal biology in humans and animals of pain states generated by persistent small afferent input

Behavioral models indicate that persistent small afferent input, as generated by tissue injury, results in a hyperalgesia at the site of injury and a tactile allodynia in areas adjacent to the injury site. Hyperalgesia reflects a sensitization of the peripheral terminal and a central facilitation evoked by the persistent small afferent input. The allodynia reflects a central sensitization. The spinal pharmacology of these pain states has been defined in the unanesthetized rat prepared with spinal catheters for injection and dialysis. After tissue injury, excitatory transmitters (e.g., glutamate and substance P) acting though N-methyl-d-aspartate (NMDA) and neurokinin 1 receptors initiate a cascade that evokes release of (i) NO, (ii) cyclooxygenase products, and (iii) activation of several kinases. Spinal dialysis show amino acid and prostanoid release after cutaneous injury. Spinal neurokinin 1, NMDA, and non-NMDA receptors enhance spinal prostaglandin E2 release. Spinal prostaglandins facilitate release of spinal amino acids and peptides. Activation by intrathecal injection of receptors on spinal C fiber terminals (μ,/∂ opiate, α2 adrenergic, neuropeptide Y) prevents release of primary afferent peptides and spinal amino acids and blocks acute and facilitated pain states. Conversely, consistent with their role in facilitated processing, NMDA, cyclooxygenase 2, and NO synthase inhibitors act to diminish only hyperalgesia. Importantly, spinal delivery of several of these agents diminishes human injury pain states. This efficacy emphasizes (i) the role of facilitated states in humans, (ii) shows the importance of spinal systems in human pain processing, and (iii) indicates that these preclinical mechanisms reflect processes that regulate the human pain experience.

Purification and Characterization of NAD-Isocitrate Dehydrogenase from Chlamydomonas reinhardtii1

NAD-isocitrate dehydrogenase (NAD-IDH) from the eukaryotic microalga Chlamydomonas reinhardtii was purified to electrophoretic homogeneity by successive chromatography steps on Phenyl-Sepharose, Blue-Sepharose, diethylaminoethyl-Sephacel, and Sephacryl S-300 (all Pharmacia Biotech). The 320-kD enzyme was found to be an octamer composed of 45-kD subunits. The presence of isocitrate plus Mn2+ protected the enzyme against thermal inactivation or inhibition by specific reagents for arginine or lysine. NADH was a competitive inhibitor (Ki, 0.14 mm) and NADPH was a noncompetitive inhibitor (Ki, 0.42 mm) with respect to NAD+. Citrate and adenine nucleotides at concentrations less than 1 mm had no effect on the activity, but 10 mm citrate, ATP, or ADP had an inhibitory effect. In addition, NAD-IDH was inhibited by inorganic monovalent anions, but l-amino acids and intermediates of glycolysis and the tricarboxylic acid cycle had no significant effect. These data support the idea that NAD-IDH from photosynthetic organisms may be a key regulatory enzyme within the tricarboxylic acid cycle.

Comparison of circumsporozoite proteins from avian and mammalian malarias: biological and phylogenetic implications.

The circumsporozoite (CS) protein of malaria parasites (Plasmodium) covers the surface of sporozoites that invade hepatocytes in mammalian hosts and macrophages in avian hosts. CS genes have been characterized from many Plasmodium that infect mammals; two domains of the corresponding proteins, identified initially by their conservation (region I and region II), have been implicated in binding to hepatocytes. The CS gene from the avian parasite Plasmodium gallinaceum was characterized to compare these functional domains to those of mammalian Plasmodium and for the study of Plasmodium evolution. The P. gallinaceum protein has the characteristics of CS proteins, including a secretory signal sequence, central repeat region, regions of charged amino acids, and an anchor sequence. Comparison with CS signal sequences reveals four distinct groupings, with P. gallinaceum most closely related to the human malaria Plasmodium falciparum. The 5-amino acid sequence designated region I, which is identical in all mammalian CS and implicated in hepatocyte invasion, is different in the avian protein. The P. gallinaceum repeat region consists of 9-amino acid repeats with the consensus sequence QP(A/V)GGNGG(A/V). The conserved motif designated region II-plus, which is associated with targeting the invasion of liver cells, is also conserved in the avian protein. Phylogenetic analysis of the aligned Plasmodium CS sequences yields a tree with a topology similar to the one obtained using sequence data from the small subunit rRNA gene. The phylogeny using the CS gene supports the proposal that the human malaria P. falciparum is significantly more related to avian parasites than to other parasites infecting mammals, although the biology of sporozoite invasion is different between the avian and mammalian species.

Expression of a delta 9 14:0-acyl carrier protein fatty acid desaturase gene is necessary for the production of omega 5 anacardic acids found in pest-resistant geranium (Pelargonium xhortorum).

Anacardic acids, a class of secondary compounds derived from fatty acids, are found in a variety of dicotyledonous families. Pest resistance (e.g., spider mites and aphids) in Pelargonium xhortorum (geranium) is associated with high levels (approximately 81%) of unsaturated 22:1 omega 5 and 24:1 omega 5 anacardic acids in the glandular trichome exudate. A single dominant locus controls the production of these omega 5 anacardic acids, which arise from novel 16:1 delta 11 and 18:1 delta 13 fatty acids. We describe the isolation and characterization of a cDNA encoding a unique delta 9 14:0-acyl carrier protein fatty acid desaturase. Several lines of evidence indicated that expression of this desaturase leads to the production of the omega 5 anacardic acids involved in pest resistance. First, its expression was found in pest-resistant, but not suspectible, plants and its expression followed the production of the omega 5 anacardic acids in segregating populations. Second, its expression and the occurrence of the novel 16:1 delta 11 and 18:1 delta 13 fatty acids and the omega 5 anacardic acids were specific to tall glandular trichomes. Third, assays of the recombinant protein demonstrated that this desaturase produced the 14:1 delta 9 fatty acid precursor to the novel 16:1 delta 11 and 18:1 delta 13 fatty acids. Based on our genetic and biochemical studies, we conclude that expression of this delta 9 14:0-ACP desaturase gene is required for the production of omega 5 anacardic acids that have been shown to be necessary for pest resistance in geranium.

Brush border myosin-I truncated in the motor domain impairs the distribution and the function of endocytic compartments in an hepatoma cell line.

Myosins I, a ubiquitous monomeric class of myosins that exhibits actin-based motor properties, are associated with plasma and/or vesicular membranes and have been suggested as players for trafficking events between cell surface and intracellular membranous structures. To investigate the function of myosins 1, we have transfected a mouse hepatoma cell line (BWTG3) with cDNAs encoding the chicken brush border myosin-I (BBMI) and two variants truncated in the motor domain. One variant is deleted of the first 446 amino acids and thereby lacks the ATP binding site, whereas the other is deleted of the entire motor domain and lacks the ATP and actin binding sites. We have observed (i) that significant amounts of the truncated variants are recovered with membrane fractions after cell fractionation, (ii) that they codistribute with a compartment containing alpha2-macroglobulin internalized for 30 min as determined by fluorescent microscopy, (iii) that the production of BBMI-truncated variants impairs the distribution of the acidic compartment and ligands internalized for 30 min, and (iv) that the production of the truncated variant containing the actin binding site decreases the rate of alpha2-macroglobulin degradation whereas the production of the variant lacking the ATP binding site and the actin binding site increases the rate of a2-macroglobulin degradation. These observations indicate that the two truncated variants have a dominant negative effect on the distribution and the function of the endocytic compartments. We propose that an unidentified myosin-I might contribute to the distribution of endocytic compartments in a juxtanuclear position and/or to the regulation of the delivery of ligands to the degradative compartment in BWTG3 cells.