Suppression of coenzyme Q10 levels and the induction of multiple PDSS and COQ genes in human cells following oligomycin treatment.

Endogenous coenzyme Q10 (CoQ10) is a lipid-soluble antioxidant and essential for the electron transport chain. We previously demonstrated that hydrogen peroxide enhanced CoQ10 levels, whereas disruption of mitochondrial membrane potential by a chemical uncoupler suppressed CoQ10 levels, in human 143B cells. In this study, we investigated how CoQ10 levels and expression of two PDSS and eight COQ genes were affected by oligomycin, which inhibited ATP synthesis at Complex V without uncoupling the mitochondria. We confirmed that oligomycin increased the production of reactive oxygen species (ROS) and decreased mitochondria-dependent ATP production in 143B cells. We also demonstrated that CoQ10 levels were decreased by oligomycin after 42 or 48 h of treatment, but not at earlier time points. Expression of PDSS2 and COQ2-COQ9 were up-regulated after 18-hour oligomycin treatment, and the expression of PPARGC1A (PGC1-1α) elevated concurrently. Knockdown of PPARGC1A down-regulated the basal mRNA levels of PDSS2 and five COQ genes and suppressed the induction of COQ8 and COQ9 genes by oligomycin, but did not affect CoQ10 levels under these conditions. N-acetylcysteine suppressed the augmentation of ROS levels and the enhanced expression of COQ2, COQ4, COQ7, and COQ9 induced by oligomycin, but did not modulate the changes in CoQ10 levels. These results suggested that the condition of mitochondrial dysfunction induced by oligomycin decreased CoQ10 levels independent of oxidative stress. Up-regulation of PDSS2 and several COQ genes by oligomycin might be regulated by multiple mechanisms, including the signaling pathways mediated by PGC-1α and ROS, but it would not restore CoQ10 levels.

Crystal structure of the kinase domain of human vascular endothelial growth factor receptor 2: a key enzyme in angiogenesis.

BACKGROUND: Angiogenesis is involved in tumor growth, macular degeneration, retinopathy and other diseases. Vascular endothelial growth factor (VEGF) stimulates angiogenesis by binding to specific receptors (VEGFRs) on the surface of vascular endothelial cells. VEGFRs are receptor tyrosine kinases that, like the platelet-derived growth factor receptors (PDGFRs), contain a large insert within the kinase domain. RESULTS: We report here the generation, kinetic characterization, and 2.4 A crystal structure of the catalytic kinase domain of VEGF receptor 2 (VEGFR2). This protein construct, which lacks 50 central residues of the 68-residue kinase insert domain (KID), has comparable kinase activity to constructs containing the entire KID. The crystal structure, determined in an unliganded phosphorylated state, reveals an overall fold and catalytic residue positions similar to those observed in other tyrosine-kinase structures. The kinase activation loop, autophosphorylated on Y1059 prior to crystallization, is mostly disordered; however, a portion of it occupies a position inhibitory to substrate binding. The ends of the KID form a beta-like structure, not observed in other known tyrosine kinase structures, that packs near to the kinase C terminus. CONCLUSIONS: The majority of the VEGFR2 KID residues are not necessary for kinase activity. The unique structure observed for the ends of the KID may also occur in other PDGFR family members and may serve to properly orient the KID for signal transduction. This VEGFR2 kinase structure provides a target for design of selective anti-angiogenic therapeutic agents.

Ionizing radiation acts on cellular membranes to generate ceramide and initiate apoptosis.

Recent investigations provided evidence that the sphingomyelin signal transduction pathway mediates apoptosis for tumor necrosis factor alpha (TNF-alpha) in several hematopoietic and nonhematopoietic cells. In this pathway, TNF-receptor interaction initiates sphingomyelin hydrolysis to ceramide by a sphingomyelinase. Ceramide acts as a second messenger stimulating a ceramide-activated serine/threonine protein kinase. The present studies show that ionizing radiation, like TNF, induces rapid sphingomyelin hydrolysis to ceramide and apoptosis in bovine aortic endothelial cells. Elevation of ceramide with exogenous ceramide analogues was sufficient for induction of apoptosis. Protein kinase C activation blocked both radiation-induced sphingomyelin hydrolysis and apoptosis, and apoptosis was restored by ceramide analogues added exogenously. Ionizing radiation acted directly on membrane preparations devoid of nuclei, stimulating sphingomyelin hydrolysis enzymatically through a neutral sphingomyelinase. These studies provide the first conclusive evidence that apoptotic signaling can be generated by interaction of ionizing radiation with cellular membranes and suggest an alternative to the hypothesis that direct DNA damage mediates radiation-induced cell kill.

Activation of the sphingomyelin signaling pathway in intact EL4 cells and in a cell-free system by IL-1 beta.

The mechanism of interleukin-1 (IL-1) signaling is unknown. Tumor necrosis factor-alpha uses a signal transduction pathway that involves sphingomyelin hydrolysis to ceramide and stimulation of a ceramide-activated protein kinase. In intact EL4 thymoma cells, IL-1 beta similarly stimulated a rapid decrease of sphingomyelin and an elevation of ceramide, and enhanced ceramide-activated protein kinase activity. This cascade was also activated by IL-1 beta in a cell-free system, demonstrating tight coupling to the receptor. Exogenous sphingomyelinase, but not phospholipases A2, C, or D, in combination with phorbol ester replaced IL-1 beta to stimulate IL-2 secretion. Thus, IL-1 beta signals through the sphingomyelin pathway.

Heterologous expression and purification of active human phosphoribosylglycinamide formyltransferase as a single domain.

We report here for the first time that the GART domain of the human trifunctional enzyme possessing GARS, AIRS, and GART activities can be expressed independently in Escherichia coli at high levels as a stable protein with enzymatic characteristics comparable to those of native trifunctional protein. Human trifunctional enzyme is involved in de novo purine biosynthesis, and has long been recognized as a target for antineoplastic intervention. The GART domain was expressed in E. coli under the control of bacteriophage T7 promotor and isolated by a three-step chromatographic procedure. Two residues, Asp 951 and His 915, were shown to be catalytically crucial by site-directed mutagenesis and subsequent characterization of purified mutant proteins. The active monofunctional GART protein produced in E. coli can serve as a valuable substitute of trifunctional enzyme for structural and functional studies which have been until now hindered because of insufficient quantity, instability, and size of the trifunctional GART protein.

Structures of apo and complexed Escherichia coli glycinamide ribonucleotide transformylase.

The three-dimensional structure of phosphoribosylglycinamide formyltransferase (10-formyltetrahydrofolate:5'-phosphoribosylglycinamide formyltransferase, EC 2.1.2.2) has been solved both as an apoenzyme at 2.8-A resolution and as a ternary complex with the substrate glycinamide ribonucleotide and a folate inhibitor at 2.5-A resolution. The structure is a modified doubly wound alpha/beta sheet with flexibility in the active site, including a disordered loop in the apo structure, which is ordered in the ternary complex structure. This enzyme is a target for anti-cancer therapy and now for structure-based drug design.

A synthetic ceramide analog, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, selectively inhibits adherence during macrophage differentiation of human leukemia cells.

Prior studies have demonstrated that sphingomyelin synthesis is involved in adherence during macrophage differentiation of HL-60 cells (Dressler, K. A., Kan, C.-C., and Kolesnick, R. N. (1991) J. Biol. Chem. 266, 11522-11527). The present studies show that glycosphingolipid synthesis is also involved. Initial studies demonstrated that the potent phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulates a 6- and 12-fold increase in the levels of sialosyllactosylceramide (GM3) and glucosylceramide (GlcCer), respectively, during development of an adherent macrophage population. In contrast, the level of lactosylceramide (LacCer) decreased to 20% of unstimulated controls. These effects were specific to adherent macrophages as nonadherent cells had minimal alteration in the glycosphingolipid profile. D-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), a competitive inhibitor of UDP-glucose:ceramide glucosyltransferase (EC 2.4.1.80), selectively blocked adherence during macrophage differentiation. PDMP markedly reduced basal levels of GlcCer, LacCer, and GM3 and prevented TPA-stimulated effects. PDMP (0.03-10 microM) reduced adherence after TPA from 30 to 6% of the total population. PDMP did not block other aspects of phorbol ester-induced macrophage differentiation including growth inhibition, expression of mRNA transcripts for the proto-oncogenes c-fos and c-myc, development of the specific enzyme markers alpha-naphthyl acetate esterase and acid phosphatase, and the gross morphologic changes associated with macrophage differentiation. PDMP appeared to have no short term or prolonged toxic effect on HL-60 cells. These studies show that PDMP selectively blocked adherence of HL-60 cells during phorbol ester-induced macrophage differentiation and suggest that this compound may be useful in the description of the biologic roles of glycosphingolipids.

Sphingomyelin synthesis is involved in adherence during macrophage differentiation of HL-60 cells.

Prior studies demonstrated that sphingomyelin degradation via a sphingomyelinase antagonized phorbol ester-mediated differentiation of HL-60 cells into macrophages (Kolesnick, R.N. (1989) J. Biol. Chem. 264, 7617-7623). The present studies show that phorbol esters induce early sphingomyelin synthesis in HL-60 cells and that this event may play a direct role in development of an adherent macrophage population. A maximally effective concentration of the potent phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA; 1 x 10(-7) M) stimulated an elevation in the sphingomyelin level at 24 h from 560 to 700 pmol/10(6) cells; a peak level of 1400 pmol/10(6) cells was achieved at 48 h. Phosphatidylcholine levels did not change significantly, indicating sphingomyelin synthesis was selective. The phosphatidylcholine:sphingomyelin ratio decreased from 10.3 to 7.9 at 24 h and to 5.3 at 48 h. Phorbol ester-induced sphingomyelin synthesis was biphasic. A burst of synthesis, detectable within 1 h and linear for 4 h, was followed by a prolonged phase at a slower rate. Ceramide synthesis was also biphasic. Ceramide levels decreased initially consistent with activation of the enzyme, phosphatidylcholine:ceramide cholinephosphotransferase and increased during the prolonged phase of sphingomyelin synthesis. During phorbol ester-induced differentiation, an adherent macrophage population was demonstrable by 14 h. This population contained the entire elevation of sphingomyelin levels. This demonstrates that early sphingomyelin synthesis defines a population of cells destined to become adherent macrophages. Studies were performed to directly manipulate sphingomyelin levels. Small unilamellar vesicles containing sphingomyelin did not directly induce macrophage differentiation but rather potentiated the effect of submaximal concentrations of phorbol ester. Sphingomyelin vesicles (2 x 10-6 M) enhanced TPA (5 x 10-10 M)-induced adherence 2-fold from 12 to 24% of the total population. Sphingosylphosphorylcholine (5 x 10-6 M), which may be acylated to sphingomyelin, was similarly effective. Further, exogenous sphingomyelinase, but not various phospholipases A2 and C, induced detachment of adherent macrophages. In sum, these studies demonstrate that phorbol esters induce early, selective synthesis of sphingomyelin in HL-60 cells. This event defines a population of cells destined to become adherent macrophages and may play a direct role in the adherence process.

Sequence of rat liver alpha 2-macroglobulin and acute phase control of its messenger RNA.

Six alpha 2-macroglobulin cDNA clones were isolated from two liver cDNA libraries produced from rats undergoing acute inflammation. The coding sequence for rat alpha 2-macroglobulin including its 27-residue signal peptide and the 3' - and part of the 5' nontranslated regions were determined. The mature protein consisting of 1445 amino acids is coded for by a 4790 +/- 40 nucleotide messenger RNA. It contains a typical internal thiol ester region and 25 cysteine residues which are conserved between rat and human alpha 2-macroglobulin. Although the amino acid sequences of rat and human alpha 2-macroglobulin share 73% identity, two small divergent areas of 17 and 38 residues were found, corresponding to 29 and 11% identity, respectively. These areas are located in the bait region and, therefore, may confer specific proteinase recognition capabilities on rat alpha 2-macroglobulin. Following an inflammatory stimulation, rat alpha 2-macroglobulin mRNA levels increased 214-fold over control values and reached a maximum at 18 h. By 24 h the levels had decreased to less than 30% of the maximum value. Transcription rates from the alpha 2-macroglobulin gene as measured in nuclear run-on experiments showed a less than 3-fold increase in nuclei from acutely inflamed rats as compared to controls. These results suggest that the accummulation of alpha 2M mRNA is due to the combined effects of increased transcription rates and post-transcriptional processing.

Nucleotide sequence of cDNA encoding human alpha 2-macroglobulin and assignment of the chromosomal locus.

Six alpha 2-macroglobulin (alpha 2M) cDNA clones were isolated from a human liver cDNA library by using synthetic oligonucleotides as hybridization probes. One of these, p alpha 2M1, carries a 4.6 kilobase-pair insert, which was sequenced. The insert contains the coding sequences for the mature alpha 2M polypeptide (1451 amino acids) and for a 23-amino acid signal peptide at the NH2 terminus of the precursor pro-alpha 2M. At the 3' end of the insert a poly(A) addition signal A-A-T-A-A-A and part of the poly(A) tail of the messenger RNA were found. The protein sequence deduced from the nucleotide sequence agrees with the published alpha 2M amino acid sequence for all except three residues. The alpha 2M locus was assigned to human chromosome 12 by Southern blot analysis with DNA from a panel of mouse/human somatic cell hybrids, using alpha 2M cDNA as a hybridization probe.

Nucleotide sequence of cDNA and derived amino acid sequence of human complement component C9.

The nucleotide sequence coding for the ninth component of human complement (C9) has been determined and the corresponding amino acid sequence has been derived. A human liver cDNA library was screened by the colony-hybridization technique using two radiolabeled oligonucleotide probes that correspond to known regions of the C9 amino acid sequence. Two recombinant plasmids were isolated and their cDNA inserts were sequenced. The derived protein sequence consists of 537 amino acids in a single polypeptide chain. A profile of the hydropathic index versus sequence number indicates that the amino-terminal half of C9 is predominantly hydrophilic in character whereas the carboxyl-terminal section of this protein is more hydrophobic. The amphipathic organization of the primary structure of C9 is consistent with the known potential of polymerized C9 to penetrate lipid bilayers, causing the formation of transmembrane channels.