Human complement subcomponent C2: purification and proteolytic cleavage in fluid phase by C1s, C1r2-C1s2 and C1

Photosynthetic membranes contain considerable regions of high surface curvature, notably at their margins, where the average radius of curvature is about 10 nm. The proportion of total membrane lipid in the outer and inner thylakoid margin monolayers is estimated at 21% and 13%, respectively. The major thylakoid lipid, monogalactosyldiacylglycerol, is roughly cone-shaped and will not form complete lamellar bilayer phases, even in combination with other thylakoid lipids. It is proposed that this galactolipid plays a role in: (a) stabilising regions of concave curvature in thylakoids; and (b) packaging hydrophobic proteins in planar bilayer regions by means of inverted micelles. This model predicts substantial asymmetries in the distribution of lipids both across and along the thylakoid bilayer plane.     

The long N-terminus of the C. elegans DNA repair enzyme APN-1 targets the protein to the nucleus of a heterologous system

We previously isolated from a Caenorhabditis elegans cDNA library, designed for two-hybrid screening, a gene encoding the DNA repair enzyme APN-1 using cross-specie complementation analysis of the Saccharomyces cerevisiae apn1? apn2? tpp1? triple mutant deficient in the ability to repair several types of DNA lesions including apurinic/apyrimidinic (AP) sites. We subsequently purified the APN-1 from this yeast mutant and demonstrated that it possesses four distinct DNA repair activities. However, following the re-annotation of the C. elegans genome we discovered that the functionally active APN-1 encoded by the cDNA from the library might lack 108 amino acid residues from the N-terminus. We therefore synthesized the entire C. elegans apn-1 gene encoding the putative full-length APN-1 and created several N-terminal deletion mutants lacking either 63, 83 or 118 amino acid residues. The full-length APN-1, APN-1 (1–63Δ) and APN-1 (1–83Δ), but not APN-1 (1–118Δ) were stably expressed in the yeast triple mutant and cleaved the AP site substrate. However, only the full-length APN-1 rescued the yeast mutant from the genotoxicity caused by methyl methane sulfonate, a DNA damaging agent that creates AP sites in the genome. The full-length APN-1 was localized to the yeast nucleus, while APN-1 (1–63Δ) and APN-1 (1–83Δ) retained a cytoplasmic distribution. Our data suggest that the N-terminal region has no direct role in the DNA repair functions of APN-1 other than to target the protein to the nucleus and possibly to maintain its stability. Thus, the truncated APN-1, previously isolated from the two-hybrid library, ability to complement the yeast triple mutant depends on the engineered SV40 nuclear localization signal.     

sNASP, a histone H1-specific eukaryotic chaperone dimer that facilitates chromatin assembly

NASP has been described as a histone H1 chaperone in mammals. However, the molecular mechanisms involved have not yet been characterized. Here, we show that this protein is not only present in mammals but is widely distributed throughout eukaryotes both in its somatic and testicular forms. The secondary structure of the human somatic version consists mainly of clusters of α-helices and exists as a homodimer in solution. The protein binds nonspecifically to core histone H2A-H2B dimers and H3-H4 tetramers but only forms specific complexes with histone H1. The formation of the NASP-H1 complexes is mediated by the N-and C-terminal domains of histone H1 and does not involve the winged helix domain that is characteristic of linker histones. In vitro chromatin reconstitution experiments show that this protein facilitates the incorporation of linker histones onto nucleosome arrays and hence is a bona fide linker histone chaperone.     

Biosynthesis and processing of pro-C3, a precursor of the third component of complement in rat hepatocytes: effect of secretion-blocking agents

Biosynthesis and intracellular processing of the third component (C3) of complement were studied in cultured rat hepatocytes. In the control cells, the complement C3 was synthesized as a pro-form, a single polypeptide chain comprising both the alpha- and beta-subunits. Although the cleavage of the pro-form into the subunits was not clearly demonstrable within the cells during pulse-chase periods, all the secreted C3 was the mature processed form. The cells were treated with secretion-blocking agents with different modes of action, colchicine and monensin. Colchicine caused an accumulation of the processed C3 within the cells, whereas monensin blocked the secretion without a significant accumulation of the processed form. The results indicate that the conversion of the C3 pro-form into the subunits takes place in the secretory vesicles just before the secretion.     

Synergistic stimulatory effect of glucocorticoid,EGF and insulin on the synthesis of ribosomal RNA and phosphorylation of nucleolin in primary cultured rat hepatocytes

Effects of dexamethasone, EGF and insulin on the synthesis of rRNA and phosphorylation of nucleolin in primary cultures of adult rat hepatocytes were studied. Hepatocytes were incubated for 8 h with EGF (20 ng/ml) plus insulin (0.1 μM) and/or for 20 h with dexamethasone (1 μM) before the end of incubation. The incorporation of [³H]uridine into acid-insoluble materials and the nuclear activity of RNA polymerase I were stimulated approx. 2-fold with EGF plus insulin and these were further enhanced 2–3-times by dexamethasone, although dexamethasone alone exerted no stimulation. When hepatocytes were incubated with [³²P]orthophosphate, similar enhancement by these hormones was also observed in the phosphorylation of a nucleolar protein, nucleolin, which was detected by immunoprecipitation with anti-nucleolin antibodies. The amount of nucleolin was slightly increased by EGF plus insulin in the presence of dexamethasone, but scarcely changed by treatment with EGF plus insulin or dexamethasone alone. Cycloheximide inhibited RNA synthesis to a greater or lesser degree in the case of all hepatocytes which were cultured with or without these hormonal treatments. These results indicate that the in vivo effect of glucocorticoid on rRNA synthesis and nucleolin phosphorylation in liver is primarily a direct action on parenchymal cells and requires other growth factors such as EGF and insulin.     

Nuclear-encoded chloroplast RNA polymerase sigma factor SIG2 activates chloroplast-encoded phycobilisome genes in a red alga, Cyanidioschyzon merolae

The phycobilisome (PBS) is a photosynthetic light-harvesting complex in red algae, whose structural genes are separately encoded by both the nuclear and chloroplast genomes. While the expression of PBS genes in both genomes is responsive to environmental changes to modulate light-harvesting efficiency, little is known about how gene expression of the two genomes is coordinated. In this study, we focused on the four nuclear-encoded chloroplast sigma factors to understand aspects of this coordination, and found that SIG2 directs the expression of chloroplast PBS genes in the red alga Cyanidioschyzon merolae.     

Inter-alpha-trypsin-inhibitor (ITI): two distinct mRNAs in baboon liver argue for a discrete synthesis of ITI and ITI derivatives

Human serum inter-alpha-trypsin-inhibitor (ITI) has so far been assumed to be comprised of a single polypeptide chain which can undergo fragmentation, whereby inhibitory ITI derivatives are released into the blood stream. In contrast, the analysis of the baboon liver mRNA translation products showed that ITI is made up of heavy and light chain(s). The latter may be excreted independently and very likely corresponds to the so-called ITI derivatives.     

Purification and molecular properties of 3 polypeptides released from a highly active O2-evolving photosystem-II preparation by Tris-treatment

Tris-treatment of a highly active O₂-evolving photosystem-II preparation induced release of 3 polypeptides (M_r 33 000, 24 000 and 18 000), concomitant with inhibition of O₂ evolution [FEBS Lett. (1981)_133. 265-268]. The 3 polypeptides were purified with the use of electrofocusing. Isoelectric points of the proteins were 5.1, 6.5 and 9.2 in order of decreasing M_r value. Only a trace amount of histidine, cystein and methionine were detected in these proteins. Based on the amino acid compositions, polarity indexes of the proteins were calculated to be 47–49%, suggesting the 3 proteins to be hydrophilic.     

C1q,a collagen-like complement subcomponent,in dermatosparactic cattle: its extracellular modification is not affected by lack of procollagen N-terminal proteinase (pN-proteinase)

C1q, a collagen-like complement protein, was purified from the serum of a ddermatosparactic calf which lacks procollagen N-terminal proteinase (pN-proteinase). The specific hemolytic activity of the serum Clq from the dermatosparactic animal was identical to that of C1q from a normal calf. Gel-filtration of serum from dermatosparactic calf, on Sepharose 6B, showed the presence of C1q-antigenic material at only one position which was identical to the elution position of normal bovine C1q. No differdence, under dissociating conditions, could be seen in the size of the chains of C1q in specific immunoprecipitates isolated from the sera of dermatosparactic and normal animals, as judged by polyacrylamidegel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). The C1q from the dermatosparactic animal showed the same N-terminal amino acid and typtic-digest peptide pattern on HPLC as C1q from the normal calf. These results strongly suggest that pN-proteinase is not involved in the extracellular processing of C1q.     

Exposure to novel environment is characterized by an interaction of D1/NMDA receptors underlined by phosphorylation of the NMDA and AMPA receptor subunits and activation of ERK1/2 signaling, leading to epigenetic changes and gene expression in rat hippocampus

Interactions between dopamine and glutamate receptors are essential for prefrontal cortical (PFC) and hippocampal cognitive functions. The hippocampus has been identified as a detector of a novel stimulus, where an association between incoming information and stored memories takes place. Further to our previous results which showed a strong synergistic interaction of dopamine D1 and glutamate NMDA receptors, the present study is going to investigate the functional status of that interaction in rats, following their exposure to a novel environment. Our results showed that the “spatial” novelty induced in rat hippocampus and PFC (a) a significant increase in phosphorylation of NMDA and AMPA receptor subunits, as well as a robust phosphorylation/activation of ERK1/2 signaling, which are both dependent on the concomitant stimulation of D1/NMDA receptors and are both abolished by habituation procedure, (b) chromatin remodeling events (phosphorylation-acetylation of histone H3) and (c) an increase in the immediate early genes (IEGs) c-Fos and zif-268 expression in the CA1 region of hippocampus, which is dependent on the co-activation of D1/NMDA and acetylcholine muscarinic receptors. In conclusion, our results clearly show that a strong synergistic interaction of D1/NMDA receptor is required for the novelty-induced phosphorylation of NMDA and AMPA receptor subunits and for the robust activation of ERK1/2 signaling, leading to chromatin remodeling events and the expression of the IEGs c-Fos and zif-268, which are involved in the regulation of synaptic plasticity and memory consolidation.     

Avian kidney mitochondrial hemeprotein P-4501α: isolation,characterization and NADPH-ferredoxin reductase-dependent activity

We describe the isolation of cytochrome P-450_1α from chick-kidney mitochondria. Although, gel permeation HPLC yielded 41% of the total amount of P-450 present in cholate-solubilized hemeproteins, it produced a highly purified mixture from which the P-450_1α could be purified to homogeneity in a final detergent-free state by a single-step application of hydrophobic interaction HPLC using hydroxypropyl silica. The purified P-450_1α traveled as a single band in SDS gel electrophoresis with an apparent M_r = 57 000. The absolute spectrum of the P-450_1α(Fe³⁺) form gave a λ_max at 403 nm. This characteristic lends support to the anomalous high-spin heme electron paramagnetic resonance spectrum and the heme structure of P-450_1α which we have previously reported (Ghazarian et al. (1980) J. Biol. Chem. 255, 8275–8281; Pedersen et al. (1976) J. Biol. Chem. 251, 3933–3941). In reconstitution experiments with ferredoxin-dependent NADPH-cytochrome c (P-450) reductase complexes, P-450_1α catalyzed the hydroxylation of 25-hydroxy-9,10-secocholesta-5,7,10(19)-trien-3β-ol at the C-1 position exclusively with a turnover number of 0.03 min^?1. This number is identical to that obtained from measurements of the catalytic activity in intact mitochondria, indicating that only one major species of cytochrome P-450 occurs in chick-kidney mitochondria. The complete responsiveness of cytochrome P-450 concentrations in intact mitochondria to the vitamin D status of chicks provided additional evidence that the major cytochrome P-450 species present in renal mitochondria is uniquely associated with vitamin D metabolism.     

Activation of human plasma prekallikrein by Pseudomonas aeruginosa elastase. II. Kinetic analysis and identification of scissile bondof prekallikrein in the activation

Activation of human plasma prekallikrein by a bacterial metalloendopeptidase, Pseudomonas aeruginosa elastase, was reported (Shibuya et al. (1991) Biochim. Biophys. Acta 1097, 23–27). Details of the activation process were presently studied. The activation accompanied limited proteolysis of a peptide bond inside of a disulfide bridge of prekallikrein molecule. Amino acid sequencing analysis of the newly generated amino-terminal revealed that the cleavage site was Arg₃₇₁-Ile₃₇₂ bond which is the scissile bond in the activation of prekallikrein with trypsin-type proteinases. A pentapeptide substrate, 2-aminobenzoyl-Ser-Thr-Ile-Val-4-nitrobenzylamide, which contained the amino acid sequence identical to that around the scissile bond of prekallikrein was synthesized. Pseudomonal elastase, indeed, hydrolyzed the substrate at Arg-Ile bond with the kinetic parameters of K_m = 118 μM, k_cat = 1.56/s and k_cat/K_m = 1.33 · 10⁴/s M. These results indicated that the Arg₃₇₁-Ile₃₇₂ bond was sensitive not only to trypsin-type serine proteinases, but also a bacterial metalloproteinase. Kinetic analysis of the prekallikrein activation by psuedomonal elastase, however, revealed that the activation rate was show, though the K_m values was good enough to expect an occurence of this activation in vivo (K_m = 248 nM, k = 6.8 · 10^?4/s, and k_cat/K_m = 2.7 · 10³/s M. The activation rate of prekallikrein by pseudomonal elastase in Hageman factor deficient plasma was remarkably improved when the plasma was reconstituted with purified Hageman factor molecule. From the results, a biologuical significance of the proteinase cascade in the plasma kinin generation was also indicated. The present in vitro study might support the hypothesis that the Hageman factor/kallikrein-kinin system plays an important role in bacterial infection including the pseudomonal one.     

Ca2+-dependent and phorbol ester activating phosphorylation of a 36K-dalton protein of rat basophilic leukemia cell membranes and immunoprecipitation of the phosphorylated protein with IgE-anti IgE system

Endogeneous phosphorylation of rat basophilic leukemia cell membranes was investigated. EGTA specifically inhibited the phosphorylation of a protein having an approximate molecular weight of 36,000 dalton (36K-Da protein). Phosphorylation of this protein was enhanced by phorbol-12-myristate-13-acetate in the presence of phosphatidylserine. The phosphorylated 36K-Da protein was specifically immunoprecipitated with IgE and anti IgE antibody. These results suggest that the phosphorylated 36K-Da protein is the beta-chain of the receptor for IgE and that protein kinase C is involved in the phosphorylation mechanism.     

Disulfide linkage in the coiled-coil domain of subunit H of A1AO ATP synthase from Methanocaldococcus jannaschii and the NMR structure of the C-terminal segment H85-104

The C-terminal residues 98-104 are important for structure stability of subunit H of A₁A_O ATP synthases as well as its interaction with subunit A. Here we determined the structure of the segment H_85-104 of H from Methanocaldococcus jannaschii, showing a helix between residues Lys90 to Glu100 and flexible tails at both ends. The helix-helix arrangement in the C-terminus was investigated by exchange of hydrophobic residues to single cysteine in mutants of the entire subunit H (H_I93C, H_L96C and H_L98C). Together with the surface charge distribution of H_85-104, these results shine light into the A-H assembly of this enzyme.     

A eukaryotic-type serine/threonine protein kinase StkP of Streptococcus pneumoniae acts as a dimer in vivo

Streptococcus pneumoniae carries a single Ser/Thr protein kinase gene stkP in its genome. Biochemical studies performed with recombinant StkP have revealed that this protein is a functional membrane-linked eukaryotic-type Ser/Thr protein kinase. Here, we demonstrate that the deletion of its extracellular domain negatively affects the stability of a core kinase domain. In contrast, the membrane anchored kinase domain and the full-length form of StkP were stable and capable of autophosphorylation. Furthermore, evidence is presented that StkP forms dimers through its transmembrane and extracellular domains.     

Identification of otubain 1 as a novel substrate for the Yersinia protein kinase using chemical genetics and mass spectrometry

Yersinia encodes a protein kinase, YpkA, which disrupts the actin cytoskeleton. Using an approach termed chemical genetics, we identified a 36-kDa substrate for YpkA in both J774 lysates and bovine brain cytosol. Mass spectrometry analysis identified this substrate as FLJ20113, an open reading frame that corresponds to otubain 1, a deubiquitinating enzyme implicated in immune cell clonal anergy. We demonstrate that otubain 1 is phosphorylated by YpkA in vitro and interacts with YpkA and actin in vivo. Identification of otubain 1 as a YpkA substrate suggests that regulation of immune cell anergy may be a survival mechanism for Yersinia.     

Monomer-dimer transition of the conserved N-terminal domain of the mammalian peroxisomal matrix protein import receptor, Pex14p

Pex14p is a central component of the peroxisomal matrix protein import machinery. In the recently determined crystal structure, a characteristic face consisting of conserved residues was found on a side of the conserved N-terminal domain of the protein. The face is highly hydrophobic, and is also the binding site for the WXXXF/Y motif of Pex5p. We report herein the dimerization of the domain in the isolated state. The homo-dimers are in equilibrium with the monomers. The homo-dimers are completely dissociated into monomers by complex formation with the WXXXF/Y motif peptide of Pex5p. A putative dimer model shows the interaction between the conserved face and the PXXP motif of another protomer. The model allows us to discuss the mechanism of the oligomeric transition of the full-length Pex14p modulated by the binding of other peroxins.