Alternative pathways of sterol synthesis in yeast. Use of C(27) sterol tracers to study aberrant double-bond migrations and evaluate their relative importance

Yeast produce traces of aberrant sterols by minor alternative pathways, which can become significant when normal metabolism is blocked by inhibitors or mutations. We studied sterols generated in the absence of the delta(8)-delta(7) isomerase (Erg2p) or delta(5) desaturase (Erg3p) by incubating three mutant strains of Saccharomyces cerevisiae with 5 alpha-cholest-8-en-3beta-ol, 8-dehydrocholesterol (delta(5,8) sterol), or isodehydrocholesterol (delta(6,8) sterol), together with the corresponding 3 alpha-3H isotopomer. Nine different incubations gave altogether 16 sterol metabolites, including seven delta(22E) sterols formed by action of the yeast C-22 desaturase (Erg5p). These products were separated by silver-ion high performance liquid chromatography (Ag(+)-HPLC) and identified by gas chromatography-mass spectrometry, nuclear magnetic resonance spectroscopy, and radio-Ag(+)-HPLC. When delta(8)-delta(7) isomerization was blocked, exogenous delta(8) sterol underwent desaturation to delta(5,8), delta(6,8), and delta(8,14) sterols. Formation of delta(5,8) sterol was strongly favored over delta(6,8) sterol, but both pathways are essentially dormant under normal conditions of sterol synthesis. The delta(5,8) sterol was metabolically almost inert except for delta(22) desaturation, whereas the delta(6,8) sterol was readily converted to delta(5,7), delta(5,7,9(11)), and delta(7,9(11)) sterols. The combined results indicate aberrant metabolic pathways similar to those in mammalian systems. However, delta(5,7) sterol undergoes only slight isomerization or desaturation in yeast, an observation that accounts for the lower levels of delta(5,8) and delta(5,7,9(11)) sterols in wild-type yeast compared to Smith-Lemli-Opitz individuals.     

Effects of sex hormones and their balance on the proliferation of rat vascular endothelial cells

OBJECTIVE: To investigate the adjustment of estrogen, progesterone and testosterone on the proliferation of female and male rat vascular endothelial cells (VECs) separately. METHODS: Rat lung VECs were cultured according to the block explanting method. MTT assay was used to measure the proliferation of VECs. RESULTS: 17beta-Estradiol (E(2)) at 3 x 10(-8) and 3 x 10(-7) M accelerated the proliferation of female rat VECs (p < 0.01). E(2) at 3 x 10(-9), 3 x 10(-8) and 3 x 10(-7) M accelerated the proliferation of male rat VECs (p < 0.05). Tamoxifen, the estrogen receptor antagonist, could block the effect of estrogen on the proliferation of VECs. Testosterone at 3 x 10(-8) and 3 x 10(-7) M significantly increased the proliferation of male rat VECs (p < 0.05), but had no effect on female rat VECs. Progesterone at 10(-9) and 10(-8) M had no effect on female rat VECs alone. When the ratio of E(2) to progesterone was 3/10, the proliferation of female rat VECs was accelerated (p < 0.05). When the ratio of E(2) to testosterone was 1/1, the proliferation of female rat VECs was also hastened (p < 0.05). However, when the ratio was reduced to 1/100, the hastening effect disappeared. CONCLUSION: Estrogen can speed up the proliferation of female and male rat VECs, while progesterone has no effect on female rat VECs alone. The balance of the ratio of E(2) to testosterone, E(2) to progesterone may play an important role in the proliferation of female rat VECs.     

A strategy for high-throughput assay development using leads derived from nuclear magnetic resonance-based screening

A strategy is described for the development of high-throughput screening assays against targets of unknown function that involves the use of nuclear magnetic resonance (NMR) spectroscopy. Using this approach, molecules that bind to the protein target are identified from an NMR-based screen of a library of substrates, cofactors, and other compounds that are known to bind to many proteins and enzymes. Once a ligand has been discovered, a fluorescent or radiolabeled analog of the ligand is synthesized that can be used in a high-throughput screen. The approach is illustrated in the development of a high-throughput screening assay against HI-0033, a conserved protein from Haemophilus influenzae whose function is currently unknown. Adenosine was found to bind to HI-0033 by NMR, and fluorescent analogs were rapidly identified that bound to HI-0033 in the submicromolar range. Using these fluorescent compounds, a fluorescence polarization assay was developed that is suitable for high-throughput screening and obtaining detailed structure-activity relationships for lead optimization.     

Cysteinyl-tRNA formation and prolyl-tRNA synthetase

Aminoacyl-tRNA (AA-tRNA) formation is a key step in protein biosynthesis. This reaction is catalyzed with remarkable accuracy by the AA-tRNA synthetases, a family of 20 evolutionarily conserved enzymes. The lack of cysteinyl-tRNA (Cys-tRNA) synthetase in some archaea gave rise to the discovery of the archaeal prolyl-tRNA (Pro-tRNA) synthetase, an enzyme capable of synthesizing Pro-tRNA and Cys-tRNA. Here we review our current knowledge of this fascinating process.     

Methanocaldococcus jannaschii prolyl-tRNA synthetase charges tRNA(Pro) with cysteine

Methanocaldococcus jannaschii prolyl-tRNA synthetase (ProRS) was previously reported to also catalyze the synthesis of cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) to make up for the absence of the canonical cysteinyl-tRNA synthetase in this organism (Stathopoulos, C., Li, T., Longman, R., Vothknecht, U. C., Becker, H., Ibba, M., and S?ll, D. (2000) Science 287, 479-482; Lipman, R. S., Sowers, K. R., and Hou, Y. M. (2000) Biochemistry 39, 7792-7798). Here we show by acid urea gel electrophoresis that pure heterologously expressed recombinant M. jannaschii ProRS misaminoacylates M. jannaschii tRNA(Pro) with cysteine. The enzyme is unable to aminoacylate purified mature M. jannaschii tRNA(Cys) with cysteine in contrast to facile aminoacylation of the same tRNA with cysteine by Methanococcus maripaludis cysteinyl-tRNA synthetase. Although M. jannaschii ProRS catalyzes the synthesis of Cys-tRNA(Pro) readily, the enzyme is unable to edit this misaminoacylated tRNA. We discuss the implications of these results on the in vivo activity of the M. jannaschii ProRS and on the nature of the enzyme involved in the synthesis of Cys-tRNA(Cys) in M. jannaschii.     

Presenilin 1 mutations activate gamma 42-secretase but reciprocally inhibit epsilon-secretase cleavage of amyloid precursor protein (APP) and S3-cleavage of notch

The presenilin 1 (PS1) and presenilin 2 (PS2) proteins are necessary for proteolytic cleavage of the amyloid precursor protein (APP) within its transmembrane domain. One of these cleavage events (termed gamma-secretase) generates the C-terminal end of the Abeta-peptide by proteolysis near residue 710 or 712 of APP(770). Another event (termed gamma-like or epsilon-secretase cleavage) cleaves near residue 721 at approximately 2-5 residues inside the cytoplasmic membrane boundary to generate a series of stable, C-terminal APP fragments. This latter cleavage is analogous to S3-cleavage of Notch. We report here that specific mutations in the N terminus, loop, or C terminus of PS1 all increase the production of Abeta(42) but cause inhibition of both epsilon-secretase cleavage of APP and S3-cleavage of Notch. These data support the hypothesis that epsilon-cleavage of APP and S3-cleavage of Notch are similar events. They also argue that, although both the gamma-site and the epsilon-site cleavage of APP are presenilin-dependent, they are likely to be independent catalytic events.     

Dynamics of human T-cell lymphotropic virus I (HTLV-I) infection of CD4+ T-cells

Stilianakis and Seydel (Bull. Math. Biol., 1999) proposed an ODE model that describes the T-cell dynamics of human T-cell lymphotropic virus I (HTLV-I) infection and the development of adult T-cell leukemia (ATL). Their model consists of four components: uninfected healthy CD4+ T-cells, latently infected CD4+ T-cells, actively infected CD4+ T-cells, and ATL cells. Mathematical analysis that completely determines the global dynamics of this model has been done by Wang et al. (Math. Biosci., 2002). In this note, we first modify the parameters of the model to distinguish between contact and infectivity rates. Then we introduce a discrete time delay to the model to describe the time between emission of contagious particles by active CD4+ T-cells and infection of pure cells. Using the results in Culshaw and Ruan (Math. Biosci., 2000) in the analysis of time delay with respect to cell-free viral spread of HIV, we study the effect of time delay on the stability of the endemically infected equilibrium. Numerical simulations are presented to illustrate the results.     

Identification of residues important for ligand binding of thromboxane A2 receptor in the second extracellular loop using the NMR experiment-guided mutagenesis approach

The second extracellular loop (eLP2) of the thromboxane A(2) receptor (TP) had been proposed to be involved in ligand binding. Through two-dimensional (1)H NMR experiments, the overall three-dimensional structure of a constrained synthetic peptide mimicking the eLP2 had been determined by our group (Ruan, K.-H., So, S.-P., Wu, J., Li, D., Huang, A., and Kung, J. (2001) Biochemistry 40, 275-280). To further identify the residues involved in ligand binding, a TP receptor antagonist, SQ29,548 was used to interact with the synthetic peptide. High resolution two-dimensional (1)H NMR experiments, NOESY, and TOCSY were performed for the peptide, SQ29,548, and peptide with SQ29,548, respectively. Through completed (1)H NMR assignment and by comparing the different spectra, extra peaks were observed on the NOESY spectrum of the peptide with SQ29,548, which implied the contacts between residues of eLP2 at Val(176), Leu(185), Thr(186), and Leu(187) with SQ29,548 at position H2, H7, and H8. Site-directed mutagenesis was used to confirm the possible ligand-binding sites on native human TP receptor. Each of the four residues was mutated to the residues either in the same group, with different structure or different charged. The mutated receptors were then tested for their ligand binding activity. The receptor with V176L mutant retained binding activity to SQ29,548. All other mutations resulted in decreased or lost binding activity to SQ29,548. These mutagenesis results supported the prediction from NMR experiments in which Val(176), Leu(185), Thr(186), and Leu(187) are the possible residues involved in ligand binding. This information facilitates the understanding of the molecular mechanism of thromboxane A(2) binding to the important receptor and its signal transduction.     

Insulin resistance in adipose tissue: direct and indirect effects of tumor necrosis factor-alpha

Insulin resistance is a fundamental defect that precedes the development of the full insulin resistance syndrome as well as beta cell failure and type 2 diabetes. Tumor necrosis factor-alpha (TNF-alpha), a paracrine/autocrine factor highly expressed in adipose tissues of obese animals and human subjects, is implicated in the induction of insulin resistance seen in obesity and type 2 diabetes. Here, we review several molecular aspects of adipose tissue physiology, and highlight the direct effects of TNF-alpha on the functions of adipose tissue including induction of lipolysis, inhibition of insulin signaling, and alterations in expression of adipocyte important genes through activation of NF-kappaB, as well as their pertinence to insulin sensitivity of adipocytes. We also review the ability of TNF-alpha to inhibit synthesis of several adipocyte-specific proteins including Acrp30 (adiponectin) and enhance release of free fatty acids (FFAs) from adipose tissue, and discuss how these factors may act as systemic mediators of TNF-alpha and affect whole body energy homeostasis and overall insulin sensitivity. On the basis of these mechanisms, we examine the therapeutic potential of blocking specific autocrine/paracrine signaling pathways in adipocytes, particularly those involving NF-kappaB, in the treatment of type 2 diabetes.