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X J Yang

Publications and source records attributed to X J Yang.

At least 73 records · Page 4Linked to original sources
PubMed1997

Acetylation of general transcription factors by histone acetyltransferases.

The acetylation of histones increases the accessibility of nucleosomal DNA to transcription factors [1,2], relieving transcriptional repression [3] and correlating with the potential for transcriptional activity in vivo [4 - 7]. The characterization of several novel histone acetyltransferases - including the human GCN5 homolog PCAF (p300/CBP-associated factor) [8], the transcription coactivator p300/CBP [9], and TAFII250 [10] - has provided a potential explanation for the relationship between histone acetylation and transcriptional activation. In addition to histones, however, other components of the basal transcription machinery might be acetylated by these enzymes and directly affect transcription. Here, we examine the acetylation of the basal transcriptional machinery for RNA polymerase II by PCAF, p300 and TAFII250. We find that all three acetyltransferases can direct the acetylation of TFIIEbetaand TFIIF, and we identify a preferred site of acetylation in TFIIEbeta. Human TFIIE consists of two subunits, alpha(p56) and beta(p34), which form a heterotetramer (alpha2 beta2) in solution ([11], reviewed in [12]). TFIIE enters the preinitiation complex after RNA polymerase II and TFIIF, suggesting that TFIIE may interact directly with RNA polymerase II and/or TFIIF [13,14]. In addition, TFIIE can facilitate promoter melting either in the presence or absence of TFIIH and can stimulate TFIIH-dependent phosphorylation of the carboxy-terminal domain of RNA polymerase II [15-18]. TFIIF has an essential role in both transcription initiation and elongation ([19,20], for review see [21]). We discuss the implications of the acetylation of TFIIEbetaand TFIIF for transcriptional control by PCAF, p300 and TAFII250.

Acetyl Coenzyme A↗
PubMed1997

Differential roles of p300 and PCAF acetyltransferases in muscle differentiation.

PCAF is a histone acetyltransferase that associates with p300/CBP and competes with E1A for access to them. While exogenous expression of PCAF potentiates both MyoD-directed transcription and myogenic differentiation, PCAF inactivation by anti-PCAF antibody microinjection prevents differentiation. MyoD interacts directly with both p300/CBP and PCAF, forming a multimeric protein complex on the promoter elements. Viral transforming factors that interfere with muscle differentiation disrupt this complex without affecting the MyoD-DNA interaction, indicating functional significance of the complex formation. Exogenous expression of PCAF or p300 promotes p21 expression and terminal cell-cycle arrest. Both of these activities are dependent on the histone acetyltransferase activity of PCAF, but not on that of p300. These results indicate that recruitment of histone acetyltransferase activity of PCAF by MyoD, through p300/CBP, is crucial for activation of the myogenic program.

Acetyl-CoA C-Acyltransferase↗
PubMed1996

The TAF(II)250 subunit of TFIID has histone acetyltransferase activity.

The transcription initiation factor TFIID is a multimeric protein complex composed of TATA box-binding protein (TBP) and many TBP-associated factors (TAF(II)s). TAF(II)s are important cofactors that mediate activated transcription by providing interaction sites for distinct activators. Here, we present evidence that human TAF(II)250 and its homologs in Drosophila and yeast have histone acetyltransferase (HAT) activity in vitro. HAT activity maps to the central, most conserved portion of dTAF(II)230 and yTAF(II)130. The HAT activity of dTAF(II)230 resembles that of yeast and human GCN5 in that it is specific for histones H3 and H4 in vitro. Our findings suggest that targeted histone acetylation at specific promoters by TAF(II)250 may be involved in mechanisms by which TFIID gains access to transcriptionally repressed chromatin.

Acetyltransferases↗
PubMed1996

A p300/CBP-associated factor that competes with the adenoviral oncoprotein E1A.

The adenoviral oncoprotein E1A induces progression through the cell cycle by binding to the products of the p300/CBP and retinoblastoma gene families. A new cellular p300/CBP-associated factor (P/CAF) having intrinsic histone acetylase activity has been identified that competes with E1A. Exogenous expression of P/CAF in HeLa cells inhibits cell-cycle progression and counteracts the mitogenic activity of E1A. E1A disturbs the normal cellular interaction between p300/CBP and its associated histone acetylase.

Acetyltransferases↗
PubMed1995

Kinetic characterization of channel impaired mutants of tryptophan synthase.

Tryptophan synthase, an alpha 2 beta 2 tetrameric complex, is a classic example of an enzyme that is thought to "channel" a metabolic intermediate (indole) from the active site of the alpha subunit to the active site of the beta subunit. The solution of the three-dimensional structure of the enzyme from Salmonella typhimurium provided physical evidence for a 25-A hydrophobic tunnel which connects the alpha and beta active sites (Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W., and Davies, D. R. (1988) J. Biol. Chem. 263, 17857-17871). Using rapid reaction kinetics, we have previously established that indole is indeed channeled and have identified three essential kinetic features which govern efficient channeling. In the current study we have probed the necessity of these features by using site-directed mutagenesis to alter these requirements. We now report the kinetic characterization of two mutants which contain substitutions to block or restrict the tunnel (beta C170F and beta C170W). Preliminary kinetic and structural evidence of a restricted tunnel in the beta C170W has been provided (Schlichting, I., Yang, X. W., Miles, E. W., Kim, A. Y., and Anderson, K. S. (1994) J. Biol. Chem. 269, 26591-26593). The rapid kinetic analysis of these mutant proteins shows that these mutations interfere with efficient channeling of the indole metabolite such that indole can be observed in single enzyme turnover of the physiologically relevant alpha beta reaction. In addition, the beta C170W mutant appears to be impaired in alpha beta intersubunit communication.

Binding Sites↗
PubMed1995

Further studies of the role of Ser-16 in the regulation of the activity of phenylalanine hydroxylase.

It was previously proposed that the activation of rat liver phenylalanine hydroxylase (EC 1.14.16.1) by cAMP-dependent protein kinase-mediated phosphorylation of Ser-16 is due to the introduction of the negatively charged phosphate group. To explore the validity of this proposal, we have applied site-directed mutagenesis to specifically replace Ser-16 with negatively charged amino acids, glutamic and aspartic; with polar uncharged amino acids, asparagine and glutamine; with the positively charged amino acid lysine; and with the nonpolar hydrophobic amino acid alanine. The wild-type and mutant enzymes were purified to homogeneity, and the importance of Ser-16 in the activation of phenylalanine hydroxylase was examined by comparing the state of activation of the phosphorylated form of the wild-type hydroxylase with that of the mutants. The kinetic studies carried out on the wild-type phosphorylated hydroxylase showed that all the activation could be accounted for by an increase in Vmax with no change in Km for either phenylalanine or the pterin cofactor. Replacement of Ser-16 with a negatively charged residue, glutamate of aspartate, resulted in the activation of the hydroxylase by 2- to 4-fold, whereas replacement with glutamine, asparagine, lysine, or alanine resulted in a much more modest increase. Further, lysolecithin was found to stimulate the phosphorylated hydroxylase and the mutant enzymes S16E and S16D by a factor of 6-7. In contrast, the mutants S16Q, S16N, and S16A all showed the same magnitude of activation as the wild-type with lysolecithin. Therefore, this study demonstrates that activation of the enzyme by phosphorylation of Ser-16 by cAMP-dependent protein kinase is due to the introduction of negative charge(s) and strongly suggests the involvement of electrostatic interaction between the regulatory and catalytic domains of the hydroxylase.

Amino Acid Sequence↗
PubMed1995

Ligand-mediated changes in the tryptophan synthase indole tunnel probed by nile red fluorescence with wild type, mutant, and chemically modified enzymes.

The bacterial tryptophan synthase alpha 2 beta 2 complex contains an unusual structural feature: an intramolecular tunnel that channels indole from the active site of the alpha subunit to the active site of the beta subunit 25 A away. Here we investigate the role of the tunnel in communication between the alpha and beta subunits using the polarity-sensitive fluorescent probe, Nile Red. Interaction of Nile Red in the nonpolar tunnel near beta subunit residues Cys-170 and Phe-280 is supported by studies with enzymes altered at these positions. Restricting the tunnel by enlarging Cys-170 by chemical modification or mutagenesis decreases the fluorescence of Nile Red by 30-70%. Removal of a partial restriction in the tunnel by replacing Phe-280 by Cys or Ser increases the fluorescence of Nile Red more than 2-fold. A binding site for Nile Red in this region near the pyridoxal phosphate coenzyme of the beta subunit is further supported by iodide quenching and fluorescence energy transfer experiments and by molecular modeling based on the three-dimensional structure of the alpha 2 beta 2 complex. Finally, studies using Nile Red as a sensitive probe of conformational changes in the tunnel reveal that allosteric ligands (alpha subunit) or active site ligands (beta subunit) decrease the fluorescence of Nile Red. We speculate that allosteric and active site ligands induce a tunnel restriction near Phe-280 that serves as a gate to control passage of indole through the tunnel.

Amino Acid Sequence↗
PubMed1994

Structural and kinetic analysis of a channel-impaired mutant of tryptophan synthase.

Substrate channeling is a process by which two sequential enzymes interact to transfer a metabolite (or intermediate) from one enzyme active site to the next without allowing free diffusion of the metabolite. Channeling is thought to play an important role in metabolic regulation and cellular modulation of enzymatic activities. Although there are numerous examples of sequential enzyme pairs in glycolysis and other biosynthetic pathways that are thought to exhibit channeling, this is as yet controversial. Tryptophan synthase is considered the best example for an enzyme displaying channeling behavior between subunits. Tryptophan synthase is an alpha 2 beta 2 tetrameric enzyme complex, which catalyzes the last two steps in the biosynthesis of L-tryptophan. The alpha subunit catalyzes the cleavage of indole-3-glycerol phosphate to indole and glyceraldehyde-3-phosphate; the beta subunit catalyzes the condensation of indole with serine to form tryptophan, in a reaction mediated by pyridoxal phosphate. The inability to trap free indole in the steady-state reaction and analysis of the kinetics of single turnover reactions have led to the postulate that indole may pass directly from the alpha to the beta site without diffusion through solution (Demoss, J. A. (1962) Biochim. Biophys. Acta 62, 279-293; Matchett, W. M. (1974) J. Biol. Chem. 249, 4041-4049). The crystal structure of tryptophan synthase from Salmonella typhimurium has provided additional support for substrate channeling by elucidating a 25-A hydrophobic tunnel connecting the two catalytic sites (Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W., and Davies, D. R. (1988) J. Biol. Chem. 263, 17857-17871). The structure suggests that mutation of a residue lining the tunnel to a more bulky residue might impede or block the passage of indole during catalysis thus enabling detection of indole during a single enzyme turnover. A mutant of tryptophan synthase has been prepared in which one of the residues lining the tunnel, beta Cys-170, has been replaced with a bulkier tryptophan residue. Kinetic and structural analyses of the beta C170W mutant by rapid chemical quench methods and x-ray crystallographic analysis show both the transient formation of indole and the obstruction of the tunnel, thus providing direct evidence for the substrate channeling mechanism.

Catalysis↗
PubMed1994

High-level expression and deletion mutagenesis of human tryptophan hydroxylase.

Human tryptophan hydroxylase has been expressed as a soluble and active form in Escherichia coli by fusion with an affinity tag, maltose-binding protein. The fusion protein has been purified to near homogeneity by affinity chromatography on crosslinked amylose resin. The purified fusion protein has a specific activity of 86 nmol of 5-hydroxytryptophan per min per mg of fusion protein. A series of truncation mutants have also been made to explore the domain organization of tryptophan hydroxylase. All deletion mutants were subject to affinity purification and kinetic characterization. While removal of the N-terminal 164 amino acids completely inactivates the enzyme, deletion of the first 91 residues results in a 7-fold reduction in specific activity. From the C terminus, deletion of 36, 55, or 112 amino acids abolishes the activity, whereas deletion of 19 residues decreases the specific activity by approximately 11-fold. These results are consistent with a model for tryptophan hydroxylase in which the enzyme consists of an N-terminal regulatory domain, a catalytic core, and a small C-terminal region of uncertain but important function.

ATP-Binding Cassette Transporters↗
PubMed1994

[Chemical constituents of Sageretia theezans Brongn].

Six compounds were isolated from Sageretia theezans and identified as friedeline (1), syringic acid (2), beta-sitosterol (3), daucosterol (4), gluco-syringic acid (5) and taraxerol (6) on the basis of spectral data (1H-NMR, IR, MS) and physico-chemical constants. Among them, compounds 5 and 6 were obtained from the species for the first time.

Benzoates↗
PubMed1993

A novel intersubunit repair mechanism in the tryptophan synthase alpha 2 beta 2 complex. Critical role of the beta subunit lysine 167 in intersubunit communication.

This study explores intersubunit communication in the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium. We find that an engineered mutation in the contact region between the alpha and beta subunits remarkably alters the catalytic and spectroscopic properties of the beta subunit in the alpha 2 beta 2 complex. Ligands that bind to the alpha subunit largely repair the deleterious effects of the beta subunit mutation in the alpha 2 beta 2 complex. The conserved residue chosen for mutation, beta subunit lysine 167, appears to form an ion pair with alpha subunit aspartate 56 in the crystal structure of the wild type alpha 2 beta 2 complex. Although replacement of beta subunit lysine 167 by threonine does not prevent formation of the alpha 2 beta 2 complex, this mutation reduces the rate of synthesis of L-tryptophan from L-serine and indole (beta reaction) 25-fold. Ligands that bind to the alpha subunit (indole-3-glycerol phosphate, indole-3-propanol phosphate, alpha-glycerol 3-phosphate, or potassium phosphate) largely restore the activity of the mutant alpha 2 beta 2 complex in the beta reaction. We conclude that beta subunit lysine 167 plays an important role in intersubunit communication in the alpha 2 beta 2 complex. The striking allosteric effects of alpha subunit ligands on the mutant beta subunit in the alpha 2 beta 2 complex may result from ligand-induced conformational changes in the alpha subunit that are transmitted to the beta subunit and repair the mutational defect in the beta subunit.

Amino Acid Sequence↗
PubMed1992

Threonine 183 and adjacent flexible loop residues in the tryptophan synthase alpha subunit have critical roles in modulating the enzymatic activities of the beta subunit in the alpha 2 beta 2 complex.

This study investigates the catalytic and allosteric roles of a flexible loop in the tryptophan synthase alpha 2 beta 2 complex. This loop connects helix 6 and strand 6 in the alpha subunit, an 8-fold alpha/beta barrel polypeptide. We have engineered three mutations in this disordered loop: a deletion of residues 185-187 and the replacement of threonine 183 by serine (T183S) or by alanine (T183A). Position 183 is a site of an inactivating mutation identified by Yanofsky's group (Yanofsky, C., Drapeau, G. R., Guest, J. R., and Carlton, B. C. (1967) Proc. Natl. Acad. Sci. U.S.A. 57, 296-298). The three engineered alpha subunits form stable, stoichiometric alpha 2 beta 2 complexes with the beta subunit which bind alpha and beta subunit ligands. Although changing threonine 183 to serine has little effect on the enzymatic properties, changing threonine 183 to alanine or deleting residues 185-187 results in a 50-fold reduction in the intrinsic activity of the alpha subunit alone and in the alpha site activity of the alpha 2 beta 2 complex. The latter two mutations profoundly alter the way in which the alpha subunit modulates the spectral properties and the activities of the wild-type beta subunit. These mutations also eliminate the effects of alpha subunit ligands on the beta subunit. Although the beta subunit ligand, L-serine, greatly stabilizes the wild-type alpha 2 beta 2 complex to dissociation and to proteolysis, L-serine stabilizes the T183A alpha 2 beta 2 complex weakly or not at all. Our findings suggest that the hydroxyl residue at position 183 and the adjacent residues in the alpha subunit loop play critical roles in the reciprocal communication between the alpha and beta subunits in the alpha 2 beta 2 complex. The results also help to explain how the wild-type alpha subunit or ammonium ion modulates the activities of the beta subunit.

Amino Acid Sequence↗
PubMed1992

Overexpression and purification of the separate tryptophan synthase alpha and beta subunits from Salmonella typhimurium.

To obtain high levels of expression of the free alpha and beta subunits of tryptophan synthase from Salmonella typhimurium, we have used two plasmids (pStrpA and pStrpB) that carry the genes encoding the alpha and beta subunits, respectively. The expression of each plasmid in Escherichia coli CB149 results in overproduction of each subunit. We also report new and efficient methods for purifying the individual alpha and beta subunits. Microcrystals of the beta subunit are obtained by addition of polyethylene glycol 8000 and spermine to crude bacterial extracts. This crystallization procedure is similar to methods used previously to grow crystals of the S. typhimurium tryptophan synthase alpha 2 beta 2 complex for X-ray crystallography and to purify this complex by crystallization from bacterial extracts. The results suggest that purification by crystallization may be useful for other overexpressed enzymes and multienzymes complexes. Purification of the alpha subunit utilizes ammonium sulfate fractionation, chromatography on diethylaminoethyl-Sephacel, and high-performance liquid chromatography on a Mono Q column. The purified alpha and beta subunits are more than 95% pure by the criterion of sodium dodecyl sulfate gel electrophoresis. The procedures developed can be applied to the expression and purification of mutant forms of the separate alpha and beta subunits. The purified alpha and beta subunits provide useful materials for studies of subunit association and for investigations of other properties of the separate subunits.

Amino Acid Sequence↗
PubMed1992

[Studies on the chemical constituents of Annona squamosa].

Twelve compounds were isolated from Annona squamosa. Their structures were identified as liriodenine (AS-1), moupinamide (AS-2), -(-)-kauran-16 alpha-ol-19-oic acid (AS-3), 16 beta, 17-dihydroxy-(-)-kauran-19-oic acid (AS-4), anonaine (AS-5), 16 alpha, 17-dihydroxy-(-)-kauran-19-oic acid (AS-6), (-)-isokaur-15(16)-en-17,19-dioic acid (AS-7), squamosamide (AS-8), 16 alpha-methoxy-(-)-kauran-19-oic acid (AS-9), sachanoic acid (AS-10), (-)-kauran-19-al-17-oic acid (AS-11), daucosterol (AS-12). Among them, AS-8 is a new amide, AS-9 is a new natural product.

Antineoplastic Agents↗
PubMed1992

[Studies on the analytical method of a new anti-arrhythmic guan-fu base A in rabbit plasma].

Guan-fu base A, a new antiarrhythmic alkaloid with a structure of C20-diterpenoid, was first isolated from the tuber of Aconitum coreanum (Levl.) Raipaics in China in 1966. An analytical method of GC-ECD has been established in this paper for the determination of its concentration in rabbit plasma in order to study its pharmacokinetics in preclinical pharmacology. Guan-fu base A and the internal standard alprenolol (ALP) was found to react rapidly and quantitatively with trifluoroacetic anhydride (TFAA) to produce derivatives GFA-TFAA and ALP-TFAA which were identified by GC-MSD. An optimal GC-ECD system for the separation and determination of the derivatives following the sample preparation was established. There was a wide linear range of the method from 10-2 x 10(4) ng/ml (r = 0.9984). The average recovery of Guan-fu base A from the spiked plasma was 97.52%. The coefficient of variation of between-day and within-day were less than 9% and 7%, respectively. The method was used to determine Guan-fu base A in the plasma of 15 rabbits following intravenous administration of 3 dosages of 0.4, 2 and 10 mg per kg of body weight.

Alkaloids↗
PubMed1991

An efficient site-directed mutagenesis using polymerase chain reaction.

We report here a simple and efficient method for site-directed mutagenesis using polymerase chain reaction (PCR). In constructing a new expression plasmid for the EcoRI restriction gene, we made two point mutations. While one created a new SalI site prior to the SD sequence, the other replaced Glu144 with Lys. A 1.5 kb SalI-PstI fragment isolated from pER101 was used as the template. Two 25 mer oligonucleotide primers containing the desired mutations were synthesized and used to direct PCR amplification with Taq DNA polymerase. About 0.5 microgram of the 0.49 kb fragment was obtained from 0.05 microgram of the 1.5 kb fragment by carrying out polymerase chain reaction for 30 cycles. As calculated theoretically, 99% of the product contained the desired mutations. The product was cloned into pUC19 using SalI and PstI, two of the transformed colonies were randomly chosen for sequence analysis, and both of them were shown to contain the desired mutations. Finally, the amplified fragment was cloned into pER304 to place the EcoRI (Lys144) gene directly under the control of the lambda PL promoter.

Base Sequence↗
PubMed1990

Activation of factor V during intrinsic and extrinsic coagulation. Inhibition by heparin, hirudin and D-Phe-Pro-Arg-Ch2Cl.

The validity of the hypothesis that Factor Xa activates Factor V in heparinized plasma was examined by establishing the temporal relationships between Factor V proteolysis and prothrombin consumption in plasma. Factor V was cleaved into Factor Va heavy chain (approx. 110 kDa) and an intermediate (approx. 230 kDa) 30 s after CaCl2 was added to contact-activated plasma (CAP). The larger fragment was converted into Factor V activation peptide (approx. 150 kDa) and Factor Va light chain (approx. 80 kDa) 15 s later. Heparin (approx. 0.05 microM) delayed Factor V proteolysis in CAP by at least 30 s. On supplementing CAP with 1 nM-Factor Xa or 1 nM-thrombin, Factor V was activated 15 s later. Heparin prolonged by 15 s and 45 s the time required to demonstrate Factor V activation in CAP supplemented with Factor Xa and thrombin respectively. Factor V was activated 20 s after tissue factor and CaCl2 were added to plasma, both in the absence and in the presence of approx. 0.05 microM-heparin. In contrast, hirudin and D-Phe-Pro-Arg-CH2Cl (two thrombin inhibitors more effective than heparin) delayed Factor V activation in this plasma by at least 30 s. The fragments of Factor V obtained in heparinized CAP suggest thrombin escapes inhibition and contributes to Factor V activation in that plasma. Production of Factor Va heavy chain and the 230 kDa Factor V fragment invariably preceded efficient prothrombin activation. These observations suggest that heparin, hirudin and D-Phe-Pro-Arg-CH2Cl delay Factor V activation by inhibiting thrombin. The availability of Factor Xa markedly moderates the ability of heparin to inhibit Factor V activation.

Affinity Labels↗