Tohru Yamamoto

Novel Substrate Specificity of the Histone Acetyltransferase Activity of HIV-1-Tat Interactive Protein Tip60

Tip60, originally isolated as an HIV-1-Tat interactive protein, contains an evolutionarily conserved domain with yeast silencing factors. We demonstrate here direct biochemical evidence that this domain of Tip60 has histone acetyltransferase activity. The purified recombinant effectively acetylates H2A, H3, and H4 but not H2B of core histone mixtures. This substrate specificity has not been observed among histone acetyltransferases analyzed to date. These results indicate that Tip60 is a histone acetyltransferase with a novel property, suggesting that Tip60 and its related factors may introduce a distinct alteration on chromatin.

The novel cargo Alcadein induces vesicle association of kinesin-1 motor components and activates axonal transport

Alcadeinα (Alcα) is an evolutionarily conserved type I membrane protein expressed in neurons. We show here that Alcα strongly associates with kinesin light chain (KD≈4–8 × 10−9 M) through a novel tryptophan‐ and aspartic acid‐containing sequence. Alcα can induce kinesin‐1 association with vesicles and functions as a novel cargo in axonal anterograde transport. JNK‐interacting protein 1 (JIP1), an adaptor protein for kinesin‐1, perturbs the transport of Alcα, and the kinesin‐1 motor complex dissociates from Alcα‐containing vesicles in a JIP1 concentration‐dependent manner. Alcα‐containing vesicles were transported with a velocity different from that of amyloid β‐protein precursor (APP)‐containing vesicles, which are transported by the same kinesin‐1 motor. Alcα‐ and APP‐containing vesicles comprised mostly separate populations in axons in vivo. Interactions of Alcα with kinesin‐1 blocked transport of APP‐containing vesicles and increased β‐amyloid generation. Inappropriate interactions of Alc‐ and APP‐containing vesicles with kinesin‐1 may promote aberrant APP metabolism in Alzheimers disease.

Analysis of structure-function relationships of yeast TATA box binding factor TFIID

A systematic series of N-terminal, C-terminal, and internal deletion mutants of S. cerevisiae TFIID were expressed in vitro and tested for TATA box binding and basal level transcription activities using, respectively, DNA mobility shift and in vitro transcription assays. The domains responsible for these activities were colocalized to a surprisingly large region containing C-terminal residues 63-240. This region was noted previously to contain potentially interesting structural motifs (central basic core, direct repeats, and sigma factor homology) and, more recently, to be highly conserved among TFIID from different species. Deletion mutant cotranslation studies revealed that TFIID binds DNA as a monomer. The implications of these results for TFIID structure and function are discussed.

X11 Proteins Regulate the Translocation of Amyloid β-Protein Precursor (APP) into Detergent-resistant Membrane and Suppress the Amyloidogenic Cleavage of APP by β-Site-cleaving Enzyme in Brain

X11 and X11-like proteins (X11L) are neuronal adaptor proteins whose association to the cytoplasmic domain of amyloid β-protein precursor (APP) suppresses the generation of amyloid β-protein (Aβ) implicated in Alzheimer disease pathogenesis. The amyloidogenic, but not amyloidolytic, metabolism of APP was selectively increased in the brain of mutant mice lacking X11L (Sano, Y., Syuzo-Takabatake, A., Nakaya, T., Saito, Y., Tomita, S., Itohara, S., and Suzuki, T. (2006) J. Biol. Chem. 281, 37853–37860). To reveal the actual role of X11 proteins (X11s) in suppressing amyloidogenic cleavage of APP in vivo, we generated X11 and X11L double knock-out mice and analyzed the metabolism of APP. The mutant mice showed enhanced β-site cleavage of APP along with increased accumulation of Aβ in brain and increased colocalization of APP with β-site APP-cleaving enzyme (BACE). In the brains of mice deficient in both X11 and X11L, the apparent relative subcellular distributions of both mature APP and its β-C-terminal fragment were shifted toward the detergent-resistant membrane (DRM) fraction, an organelle in which BACE is active and both X11s are not nearly found. These results indicate that X11s associate primarily with APP molecules that are outside of DRM, that the dissociation of APP-X11/X11L complexes leads to entry of APP into DRM, and that cleavage of uncomplexed APP by BACE within DRM is enhanced by X11s deficiency. Present results lead to an idea that the dysfunction of X11L in the interaction with APP may recruit more APP into DRM and increase the generation of Aβ even if BACE activity did not increase in brain.

Specific Interactions and Potential Functions of Human TAFII100

Human transcription initiation factor TFIID contains the TATA-binding protein (TBP) and several TBP-associated factors (TAFs). To investigate the structural organization and function of TFIID, we have cloned and expressed a cDNA encoding the third largest human TFIID subunit, hTAFII100. Immunoprecipitation studies demonstrate that hTAFII100 is an integral subunit that is associated with all transcriptionally-competent forms of TFIID. They further suggest that at least part of the N-terminal region lies on the surface of TFIID, while a C-terminal region containing conserved WD-40 repeats appears inaccessible. Both in vivo and in vitro assays indicate that hTAFII100 interacts strongly with the histone H4-related hTAFII80 and the histone H3-related hTAFII31, as well as a stable complex comprised of both hTAFII80 and hTAFII31. Apparently weaker interactions of hTAFII100 with TBP, hTAFII250, hTAFII28, and hTAFII20, but not hTAFII55, also have been observed. These results suggest a role for hTAFII100 in stabilizing interactions of TAFs, especially the histone-like TAFs, in TFIID. In addition, functional studies show that anti-hTAFII100 antibodies selectively inhibit basal transcription from a TATA-less initiator-containing promoter, relative to a TATA-containing promoter, suggesting a possible core promoter-specific function for hTAFII100.

Alcadein Cleavages by Amyloid β-Precursor Protein (APP) α- and γ-Secretases Generate Small Peptides, p3-Alcs, Indicating Alzheimer Disease-related γ-Secretase Dysfunction

Alcadeins (Alcs) constitute a family of neuronal type I membrane proteins, designated Alcα, Alcβ, and Alcγ. The Alcs express in neurons dominantly and largely colocalize with the Alzheimer amyloid precursor protein (APP) in the brain. Alcs and APP show an identical function as a cargo receptor of kinesin-1. Moreover, proteolytic processing of Alc proteins appears highly similar to that of APP. We found that APP α-secretases ADAM 10 and ADAM 17 primarily cleave Alc proteins and trigger the subsequent secondary intramembranous cleavage of Alc C-terminal fragments by a presenilin-dependent γ-secretase complex, thereby generating “APP p3-like” and non-aggregative Alc peptides (p3-Alcs). We determined the complete amino acid sequence of p3-Alcα, p3-Alcβ, and p3-Alcγ, whose major species comprise 35, 37, and 31 amino acids, respectively, in human cerebrospinal fluid. We demonstrate here that variant p3-Alc C termini are modulated by FAD-linked presenilin 1 mutations increasing minor β-amyloid species Aβ42, and these mutations alter the level of minor p3-Alc species. However, the magnitudes of C-terminal alteration of p3-Alcα, p3-Alcβ, and p3-Alcγ were not equivalent, suggesting that one type of γ-secretase dysfunction does not appear in the phenotype equivalently in the cleavage of type I membrane proteins. Because these C-terminal alterations are detectable in human cerebrospinal fluid, the use of a substrate panel, including Alcs and APP, may be effective to detect γ-secretase dysfunction in the prepathogenic state of Alzheimer disease subjects.

Antisense RNA of the latent period gene (MER5) inhibits the differentiation of murine erythroleukemia cells.

The MER5 cDNA was cloned from RNA preferentially synthesized in murine erythroleukemia (MEL) cells during the early period of MEL cell differentiation. To understand the role of the MER5 gene in the differentiation, we have transferred the MER5 cDNA into MEL cells in both sense and antisense orientations under control of the promoter of the human metallothionein gene. Only in the transformants with the antisense MER5 cDNA, did their elevated expression inhibit differentiation. The result suggests that the MER5 gene product may promote early events in the differentiation of MEL cells.

Cloning of the cDNA encoding a novel subtype of histone H1.

The H1 class of histones is implicated in organizing a higher order of chromatin structure and in involvement with transcriptional repression. They compromise multiple subtypes that presumably have a specific function. We report here the isolation and characterization of a human cDNA encoding a novel subtype of histone H1. It is the most distantly related subtype of mammalian H1 reported to date. However, it still shares the characteristic features of H1: the tripartite structure, conservation in the globular domain, the profile of hydropathy and the basic isoelectric point. The expression of this H1 subtype was ubiquitously observed in all tissues examined. Our findings suggest that novel subtypes of H1 may remain unidentified in mammals.

The conserved carboxy-terminal domain of Saccharomyces cerevisiae TFIID is sufficient to support normal cell growth.

We have examined the structure-function relationships of TFIID through in vivo complementation tests. A yeast strain was constructed which lacked the chromosomal copy of SPT15, the gene encoding TFIID, and was therefore dependent on a functional plasmid-borne wild-type copy of this gene for viability. By using the plasmid shuffle technique, the plasmid-borne wild-type TFIID gene was replaced with a family of plasmids containing a series of systematically mutated TFIID genes. These various forms of TFIID were expressed from three different promoter contexts of different strengths, and the ability of each mutant form of TFIID to complement our chromosomal TFIID null allele was assessed. We found that the first 61 amino acid residues of TFIID are totally dispensable for vegetative cell growth, since yeast strains containing this deleted form of TFIID grow at wild-type rates. Amino-terminally deleted TFIID was further shown to be able to function normally in vivo by virtue of its ability both to promote accurate transcription initiation from a large number of different genes and to interact efficiently with the Gal4 protein to activate transcription of GAL1 with essentially wild-type kinetics. Any deletion removing sequences from within the conserved carboxy-terminal region of S. cerevisiae TFIID was lethal. Further, the exact sequence of the conserved carboxy-terminal portion of the molecule is critical for function, since of several heterologous TFIID homologs tested, only the highly related Schizosaccharomyces pombe gene could complement our S. cerevisiae TFIID null mutant. Taken together, these data indicate that all important functional domains of TFIID appear to lie in its carboxy-terminal 179 amino acid residues. The significance of these findings regarding TFIID function are discussed.

Suppression of APP‐containing vesicle trafficking and production of β‐amyloid by AID/DHHC‐12 protein

The metabolism of amyloid β‐protein precursor (APP) is regulated by various cytoplasmic and/or membrane‐associated proteins, some of which are involved in the regulation of intracellular membrane trafficking. We found that a protein containing Asp–His–His–Cys (DHHC) domain, alcadein and APP interacting DHHC protein (AID)/DHHC‐12, strongly inhibited APP metabolism, including amyloid β‐protein (Aβ) generation. In cells expressing AID/DHHC‐12, APP was tethered in the Golgi, and APP‐containing vesicles disappeared from the cytoplasm. Although DHHC domain‐containing proteins are involved in protein palmitoylation, a AID/DHHC‐12 mutant of which the enzyme activity was impaired by replacing the DHHC sequence with Ala–Ala–His–Ser (AAHS) made no detectable difference in the generation and trafficking of APP‐containing vesicles in the cytoplasm or the metabolism of APP. Furthermore, the mutant AID/DHHC‐12 significantly increased non‐amyloidogenic α‐cleavage of APP along with activation of a disintegrin and metalloproteinase 17, a major α‐secretase, suggesting that protein palmitoylation involved in the regulation of α‐secretase activity. AID/DHHC‐12 can modify APP metabolism, including Aβ generation in multiple ways by regulating the generation and/or trafficking of APP‐containing vesicles from the Golgi and their entry into the late secretary pathway in an enzymatic activity‐independent manner, and the α‐cleavage of APP in the enzymatic activity‐dependent manner.