Tetsuro Kokubo

The TAFII250 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.

The Yeast TAF145 Inhibitory Domain and TFIIA Competitively Bind to TATA-Binding Protein

ABSTRACT The Drosophila 230-kDa TFIID subunit (dTAF230) interacts with the DNA binding domain of TATA box-binding protein (TBP) which exists in the same complex. Here, we characterize the inhibitory domain in the yeast TAF145 (yTAF145), which is homologous to dTAF230. Mutation studies show that the N-terminal inhibitory region (residues 10 to 71) can be divided into two subdomains, I (residues 10 to 37) and II (residues 46 to 71). Mutations in either subdomain significantly impair function. Acidic residues in subdomain II are important for the interaction with TBP. In addition, yTAF145 interaction is impaired by mutating the basic residues on the convex surface of TBP, which are crucial for interaction with TFIIA. Consistently, TFIIA and yTAF145 bind competitively to TBP. A deletion of the inhibitory domain of yTAF145 leads to a temperature-sensitive growth phenotype. Importantly, this phenotype is suppressed by overexpression of the TFIIA subunits, indicating that the yTAF145 inhibitory domain is involved in TFIIA function.

Molecular characterization of L-amino acid oxidase from Agkistrodon halys blomhoffii with special reference to platelet aggregation.

L-Amino acid oxidase (LAO, EC 1.4.3.2) is widely distributed in snake venom, and induces apoptosis in vascular endothelial cells, causing prolonged bleeding from vessel walls at bite sites. The effect of snake venom LAOs on platelet function is controversial. Further, we have little information on their structural characterization. We purified M (mamushi)-LAO, a single-chain glycoprotein with a molecular mass of 60 kDa and a pI of 4.9, from Agkistrodon halys blomhoffii (Japanese mamushi) venom, and determined the N-terminal and several internal amino acid sequences of this enzyme. Molecular cloning based on these data was conducted to elucidate its full-length cDNA structure (2192 nucleotides), which includes a putative 18 amino acid residue signal peptide and a 504 residue mature subunit. The predicted M-LAO translation product shares 87.3% identity with that of Crotalus adamanteus (Southeastern diamondback rattlesnake) LAO. M-LAO, up to a final concentration of 2.6 microM, inhibited both agonist- and shear stress-induced platelet aggregation (SIPA) dose-dependently. In agonist-induced platelet aggregation, M-LAO predominantly inhibited the second aggregation, but with a marginal inhibition of the first. In SIPA, the inhibition was more dramatic under low-shear stress than high-shear stress, and was enhanced by the presence of L-leucine, a substrate of this enzyme. Catalase, a H2O2 scavenger, totally quenched such enhancement. These results suggest that M-LAO inhibits the interaction between activated platelet integrin alphaIIb/beta3 and fibrinogen through the continuous generation of H2O2, and may contribute to prolonged bleeding from the vessels at snake bite sites.

Identification of Highly Conserved Amino-terminal Segments of dTAFII230 and yTAFII145 That Are Functionally Interchangeable for Inhibiting TBP-DNA Interactions in Vitro and in Promoting Yeast Cell Growth in Vivo

TFIID is a multiprotein complex composed of TBP and several TAFIIs. Small amino-terminal segments (TAF N-terminal domain (TAND)) of DrosophilaTAFII230 (dTAFII230) and yeast TAFII145 (yTAFII145) bind strongly to TBP and inhibit TBP-DNA interactions. yTAFII145 TAND (yTAND) was divided into two subdomains, yTANDI10–37 and yTANDII46–71, that function cooperatively. Here, we identify dTANDII within the amino terminus of dTAFII230 at 118–143 amino acids in addition to dTANDI18–77, reported previously. dTANDII exhibits pronounced sequence similarity to yTANDII, and the two were shown to be functionally equivalent in binding to TBP and inhibiting TBP-DNA interactions in vitro. Alanine scanning mutation analysis demonstrated that Phe-57 (yTANDII) and Tyr-129 (dTANDII) are critically required for the interaction with TBP. Yeast strains containing mutant yTAFII145 lacking yTANDI or yTANDII showed a temperature-sensitive growth phenotype. The conserved core of dTANDII could substitute for the yTANDII core, and Phe-57 or Tyr-129 described above was critically required for the function of this segment in promoting normal cell growth at 37u2009°C. In these respects, the impact of yTANDII mutations on cell growth paralleled their effects on TBP binding in vitro, strongly suggesting that the yTAFII145-TBP interaction and its negative effects on TFIID binding to core promoters are physiologically important.

The Saccharomyces cerevisiae RuvB-like Protein, Tih2p, Is Required for Cell Cycle Progression and RNA Polymerase II-directed Transcription

Two highly conserved RuvB-like putative DNA helicases, p47/TIP49b and p50/TIP49a, have been identified in the eukaryotes. Here, we study the function of Saccharomyces cerevisiae TIH2, which corresponds to mammalian p47/TIP49b. Tih2p is required for vegetative cell growth and localizes in the nucleus. Immunoprecipitation analysis revealed that Tih2p tightly interacts with Tih1p, the counterpart of mammalian p50/TIP49a, which has been shown to interact with the TATA-binding protein and the RNA polymerase II holoenzyme complex. Furthermore, the mutational study of the Walker A motif, which is required for nucleotide binding and hydrolysis, showed that this motif plays indispensable roles in the function of Tih2p. When a temperature-sensitive tih2 mutant,tih2–160, was incubated at the nonpermissive temperature, cells were rapidly arrested in the G1 phase. Northern blot analysis revealed that Tih2p is required for transcription of G1 cyclin and of several ribosomal protein genes. The similarities between the mutant phenotypes of tih2–160 and those of taf145 mutants suggest a role for TIH2in the regulation of RNA polymerase II-directed transcription.

Mutations in the TATA-binding Protein, Affecting Transcriptional Activation, Show Synthetic Lethality with the TAF145Gene Lacking the TAF N-terminal Domain in Saccharomyces cerevisiae

The general transcription factor TFIID, which is composed of the TATA box-binding protein (TBP) and a set of TBP-associated factors (TAFs), is crucial for both basal and regulated transcription by RNA polymerase II. The N-terminal small segment of yeast TAF145 (yTAF145) binds to TBP and thereby inhibits TBP function. To understand the physiological role of this inhibitory domain, which is designated as TAND (TAF N-terminaldomain), we screened mutations, synthetically lethal with the TAF145 gene lacking TAND (taf145ΔTAND), in Saccharomyces cerevisiae by exploiting a red/white colony-sectoring assay. Our screen yielded several recessive nsl (ΔTANDsynthetic lethal) mutations, two of which,nsl1-1 and nsl1-2, define the same complementation group. The NSL1 gene was found to be identical to the SPT15 gene encoding TBP. Interestingly, both temperature-sensitive nsl1/spt15 alleles, which harbor the single amino acid substitutions, S118L and P65S, respectively, were defective in transcriptional activation in vivo. Several other previously characterized activation-deficient spt15alleles also displayed synthetic lethal interactions withtaf145ΔTAND, indicating that TAND and TBP carry an overlapping but as yet unidentified function that is specifically required for transcriptional regulation.

The cDNA cloning of human placental ecto-ATP diphosphohydrolases I and II1

The cDNA clones of two isoforms (enzymes I and II) of human placental ecto‐ATP diphosphohydrolases have been isolated based on the N‐terminal amino acid (aa) sequence of the immunopurified 82 kDa protein and characterized. The cDNA clone encoding enzyme I consists of 2081 nucleotides and the predicted enzyme I consists of 517 aa residues. Enzyme I has a 5′‐UTR and an N‐terminal 11 aa sequence that differ from CD39, but the rest of the sequence is the same as CD39. The hydropathy plot indicated that enzyme I has two hydrophobic regions near the N‐ and C‐termini of the molecule. In contrast, enzyme II consists of 1814 nucleotides and the predicted protein consists of 306 aa residues. The sequence of 1–1018 nucleotides of enzyme II is identical to that of enzyme I, but the 1019–1814 nucleotide sequence is different from both enzyme I and CD39. The hydropathy plot indicated that enzyme II has one hydrophobic region near the N‐terminus, suggesting that enzyme II is also anchored to the cell membrane. It is, however, likely that some of enzyme II exists as a soluble form in plasma, possibly after proteolytic processing.

Total inhibition of high shear stress induced platelet aggregation by homodimeric von Willebrand factor A1-loop fragments.

Under high shear stress, the binding of von Willebrand factor (VWF) A1‐loop domain to platelet glycoprotein (GP) Ibα occurs as the earliest event in thrombus formation. Therefore recombinant VWF A1‐loop fragments could be of therapeutic use in blocking this interaction as competing ligands. We have prepared three homodimeric VWF A1‐loop fragments [315u2003kD Fr III (a homodimer of amino acid [aa] residues 1–1365 of the subunit), 220u2003kD Fr (a homodimer of aa residues 1–708 of the subunit), and 116u2003kD Fr (a homodimer of aa residues 449–728 of the subunit) and two monomeric fragments [39/34u2003kD Fr (a monomer of aa residues 480/481–718 of the subunit] and His‐rVWF465–728 (a monomer of aa residues 465–728 of the subunit)], and assessed their potency as inhibitors of botrocetin‐induced VWF binding to GPIbα and high shear stress induced platelet aggregation mediated by intact VWF. All these fragments completely inhibited botrocetin‐induced VWF binding to GPIbα at a final concentration of 40–200u2003μm. The homodimeric A1‐loop fragments also totally inhibited high shear stress induced platelet aggregation at a final concentration of 0.45–2.0u2003μm in the following order: 315u2003kD Fru2003u2003220u2003kD Fru2003≫u2003116u2003kD Fr. In contrast, the monomeric A1‐loop fragments were only partial inhibitors of high shear stress induced platelet aggregation even at a final concentration of 20u2003μm, and their IC50s were 13–39 times higher than those of the homodimers. These results indicate that the homodimeric structure of the A1‐loop fragment is important for optimal molecular interaction with GPIbα under high shear stress.