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.     

Aberrant pathways in the late stages of cholesterol biosynthesis in the rat. Origin and metabolic fate of unsaturated sterols relevant to the Smith-Lemli-Opitz syndrome

Minor aberrant pathways of cholesterol biosynthesis normally produce only trace levels of abnormal sterol metabolites but may assume major importance when an essential biosynthetic step is blocked. Cholesta-5,8-dien-3beta-ol, its Delta(5,7) isomer, and other noncholesterol sterols accumulate in subjects with the Smith-Lemli-Opitz syndrome (SLOS), a severe developmental disorder caused by a defective Delta(7) sterol reductase gene. We have explored the formation and metabolism of unsaturated sterols relevant to SLOS by incubating tritium-labeled Delta(5,8), Delta(6, 8), Delta(6,8(14)), Delta(5,8(14)), and Delta(8) sterols with rat liver preparations. More than 60 different incubations were carried out with washed microsomes or the 10,000 g supernatant under aerobic or anaerobic conditions; some experiments included addition of cofactors, fenpropimorph (a Delta(8);-Delta(7) isomerase inhibitor), and/or AY-9944 (a Delta(7) reductase inhibitor). The tritium-labeled metabolites from each incubation were identified by silver ion high performance liquid chromatography on the basis of their coelution with unlabeled authentic standards, as free sterols and/or acetate derivatives. The Delta(5,8) sterol was converted slowly to cholesterol via the Delta(5,7) sterol, which also slowly isomerized back to the Delta(5,8) sterol. The Delta(6,8) sterol was metabolized rapidly to cholesterol by an oxygen-requiring pathway via the Delta(7,9(11)), Delta(8), Delta(7), and Delta(5,7) sterols as well as by an oxygen-independent route involving initial isomerization to the Delta(5,7) sterol. The Delta(8) sterol was partially metabolized to Delta(5,8), Delta(6,8), Delta(7,9(11)), and Delta(5,7,9(11)) sterols when isomerization to Delta(7) was blocked.The combined results were used to formulate a scheme of normal and aberrant biosynthetic pathways that illuminate the origin and metabolic fate of abnormal sterols observed in SLOS and chondrodysplasia punctata.     

Estrogen receptors and the activity of nitric oxide synthase in the artery of female rats receiving hormone replacement therapy

OBJECTIVE: To observe the expression of estrogen receptor and the activity of NOS in the arteries of female rats receiving estrogen replacement therapy. METHODS: Seventy-two female rats were randomly divided into four groups: group A: sham-ovariectomy; group B: ovariectomy; group C: ovariectomy with estrogen replacement therapy (benzoate estradiol, 5 microg i.m. once in 2 days); group D: ovariectomy with estrogen and progesterone replacement therapy (benzoate estradiol, 5 microg i.m. once in 2 days and progesterone, 1 mg i.m. once in 2 days). The rats were killed after 2 months. The receptor-binding assay was adopted to measure the estrogen receptors in the arteries of the rats, and the activity of NOS in the arteries was assessed by the hemoglobin reductase method. RESULTS: The ER number and NOS activity in the arteries of the ovariectomized group are less than those in sham-ovariectomy group (p<0.05). The ER number and NOS activity in the arteries of groups C and D are larger and higher than those in the ovariectomized group (p<0.05). No significant differences in the ER number and NOS activity were observed between groups C and D. CONCLUSION: The ER number and NOS activity in the rat artery significantly decrease after ovariectomy, while hormone replacement therapy can significantly increase the artery NOS activity and retain the ER number in the artery of the ovariectomized rats to normal level. The result may contribute to explaining the beneficial effect of estrogen in the prevention of coronary artery diseases in postmenopausal women.     

Histone acetyltransferases MystA and MystB contribute to morphogenesis and aflatoxin biosynthesis by regulating acetylation in fungus Aspergillus flavus

Myst family is highly conserved histone acetyltransferases in eukaryotic cells and is known to play crucial roles in various cellular processes; however, acetylation catalysed by acetyltransferases is unclear in filamentous fungi. Here, we identified two classical nonessential Myst enzymes and analysed their functions in Aspergillus flavus, which generates aflatoxin B1, one of the most carcinogenic secondary metabolites. MystA and MystB located in nuclei and cytoplasm, and mystA could acetylate H4K16ac, while mystB acetylates H3K14ac, H3K18ac and H3K23ac. Deletion mystA resulted in decreased conidiation, increased sclerotia formation and aflatoxin production. Deletion of mystB leads to significant defects in conidiation, sclerotia formation and aflatoxin production. Additionally, double-knockout mutant (ΔmystA/mystB) display a stronger and similar defect to ΔmystB mutant, indicating that mystB plays a major role in regulating development and aflatoxin production. Both mystA and mystB play important role in crop colonization. Moreover, catalytic domain MOZ and the catalytic site E199/E243 were important for the acetyltransferase function of Myst. Notably, chromatin immunoprecipitation results indicated that mystB participated in oxidative detoxification by regulating the acetylation level of H3K14, and further regulated nsdD to affect sclerotia formation and aflatoxin production. This study provides new evidences to discover the biological functions of histone acetyltransferase in A. flavus.