Tsutomu Fujiwara

TAZ: a novel transcriptional co-activator regulated by interactions with 14-3-3 and PDZ domain proteins

The highly conserved and ubiquitously expressed 14‐3‐3 proteins regulate differentiation, cell cycle progression and apoptosis by binding intracellular phosphoproteins involved in signal transduction. By screening in vitro translated cDNA pools for the ability to bind 14‐3‐3, we identified a novel transcriptional co‐activator, TAZ (transcriptional co‐activator with PDZ‐binding motif) as a 14‐3‐3‐binding molecule. TAZ shares homology with Yes‐associated protein (YAP), contains a WW domain and functions as a transcriptional co‐activator by binding to the PPXY motif present on transcription factors. 14‐3‐3 binding requires TAZ phosphorylation on a single serine residue, resulting in the inhibition of TAZ transcriptional co‐activation through 14‐3‐3‐mediated nuclear export. The C‐terminus of TAZ contains a highly conserved PDZ‐binding motif that localizes TAZ into discrete nuclear foci and is essential for TAZ‐stimulated gene transcription. TAZ uses this same motif to bind the PDZ domain‐containing protein NHERF‐2, a molecule that tethers plasma membrane ion channels and receptors to cytoskeletal actin. TAZ may link events at the plasma membrane and cytoskeleton to nuclear transcription in a manner that can be regulated by 14‐3‐3.

Macular corneal dystrophy type I and type II are caused by distinct mutations in a new sulphotransferase gene

Macular corneal dystrophy (MCD; MIM 217800) is an autosomal recessive hereditary disease in which progressive punctate opacities in the cornea result in bilateral loss of vision, eventually necessitating corneal transplantation. MCD is classified into two subtypes, type I and type II, defined by the respective absence and presence of sulphated keratan sulphate in the patient serum, although both types have clinically indistinguishable phenotypes. The gene responsible for MCD type I has been mapped to chromosome 16q22, and that responsible for MCD type II may involve the same locus. Here we identify a new carbohydrate sulphotransferase gene (CHST6), encoding an enzyme designated corneal N-acetylglucosamine-6-sulphotransferase (C-GlcNAc6ST), within the critical region of MCD type I. In MCD type I, we identified several mutations that may lead to inactivation of C-GlcNAc6ST within the coding region of CHST6. In MCD type II, we found large deletions and/or replacements caused by homologous recombination in the upstream region of CHST6. In situ hybridization analysis did not detect CHST6 transcripts in corneal epithelium in an MCD type II patient, suggesting that the mutations found in type II lead to loss of cornea-specific expression of CHST6.

Molecular properties of the proteasome activator PA28 family proteins and γ-interferon regulation

Background: Recent cDNA cloning of two homologous proteasome activators, PA28α and PA28β, indicated the presence of a structurally related third protein, Ki antigen, but a functional relationship between Ki antigen and the two PA28 proteins is unknown. Accumulating evidence has implicated an important role for PA28 in the major histocompatibility complex (MHC) class I‐restricted antigen processing pathway. Recently, an immunomodulatory cytokine γ‐interferon (γ‐IFN) was found to increase greatly the messages for PA28α and PA28β, but not Ki antigen, in human cells.

Possible mechanism of nuclear translocation of proteasomes

Proteasomes (multicatalytic proteinase complexes), which are identical to the ubiquitous eukaryotic 20S particles, are localized in both the cytoplasm and the nucleus, but the mechanism of their co‐localization in the two compartments is unknown. On examination of the primary structures of subunits of proteasomes, a consensus sequence for nuclear translocation of proteins, X‐X‐K‐K(R)‐X‐K(R) (where X is any residue), was found to be present in some subunits and to be highly conserved in the subunits of a wide range of eukaryotes. In addition, proteasomal subunits were found to bear a cluster of acidic amino acid residues and also a potential tyrosine phosphorylation site that was located in the same polypeptide chain as the nuclear location signal. These structural properties suggest that two sets of clusters with positive and negative charges serve to regulate the translocation of proteasomes from the cytoplasm to the nucleus, and that phosphorylation of tyrosine in certain subunits may play an additional role in transfer of proteasomes into the nucleus.

Cloning and chromosomal mapping of a novel ABC transporter gene (hABC7), a candidate for X-linked sideroblastic anemia with spinocerebellar ataxia

AbstractWe isolated a novel human ATP-binding cassette (ABC) transporter cDNA, determined its nucleotide sequence, and designated it human ABC7 (hABC7). The nucleotide sequence was highly homologous to the ATM1 gene in yeast, which encodes an ABC transporter (yAtm1p) located in the mitochondrial inner membrane. The deduced human product, a putative half-type transporter, consists of 752 amino acids that are 48.9% identical to those of yAtm1p. A computer-assisted protein structural and localization analysis revealed that the mitochondrial targeting signal of yAtm1p is conserved in the N-terminal region of the primary sequence of the hABC7 protein, and therefore this product is also likely to be located in the mitochondrial inner membrane. The evidence strongly suggests that the hABC7 gene is a counterpart of ATM1 and that its product is probably involved in heme transport. We mapped the hABC7 gene to chromosome Xq13.1–q13.3 by fluorescence in-situ hybridization. As band Xq13 has been implicated in X-linked sideroblastic anemia with spinocerebellar ataxia, hABC7 becomes a candidate gene for this heritable disorder.

Isolation, mapping, and functional analysis of a novel human cDNA (BNIP3L) encoding a protein homologous to human NIP3

We have isolated a novel cDNA that encodes a product showing significant sequence homology (56% identity) to human NIP3, a protein thought to interact with adenovirus E1B19kD and human BCL2 proteins. This cDNA contains an open reading frame of 657 nucleotides encoding a 219 amino acid polypeptide. The gene, designated BNIP3L, was expressed in all 16 normal human tissues examined; we mapped it to chromosome band 8p21 by fluorescence in situ hybridization. Introduction of the BNIP3L gene into six different cancer‐cell lines caused significant growth suppression in each of them, while no such effect occurred when the antisense cDNA or the vector DNA was transfected, indicating that BNIP3L may function as a tumor suppressor. Genes Chromosomes Cancer 21:230–235, 1998.

MOLECULAR CLONING AND SEQUENCE ANALYSIS OF CDNAS FOR FIVE MAJOR SUBUNITS OF HUMAN PROTEASOMES (MULTI-CATALYTIC PROTEINASE COMPLEXES)

Proteasomes are multicatalytic proteinase complexes consisting of a set of non-identical polypeptide components. Of these multiple components, the nucleotide sequences of five major subunits (named HC2, HC3, HC5, HC8 and HC9) of human proteasomes have been determined from recombinant cDNA clones by screening a human HepG2 hepatoblastoma cell cDNA library with rat proteasome cDNAs isolated previously as probes. The polypeptides deduced from their nucleotide sequences consisted of 263, 234, 241, 255 and 261 amino acid residues with calculated molecular weights of 29,554, 25,897, 26,487, 28,431 and 29,482, respectively, which are encoded by single independent genes. The primary structures of these subunits of human proteasomes closely resemble those of their rat counterparts and show considerably high inter-subunit homology, although the homology of HC5 is relatively low. These findings, together with the structural similarities of other eukaryotic proteasomes including those of Drosophila and yeast (Saccharomyces cerevisiae) support and extend the previously proposed concept that eukaryotic proteasome genes form a multi-gene family with the same evolutionary origin.

Isolation and expression of a cDNA for human brain fatty acid-binding protein (B-FABP).

We have isolated and sequenced a novel 754-bp human fetal brain cDNA encoding a fatty acid-binding protein (FABP). The cDNA contains an open reading frame of 396 nucleotides (132 amino acids). Northern analysis revealed small amounts of a 1.2-kb transcript in adult human brain and a shorter transcript in skeletal muscle, but no message was detected in other tissues examined. On the other hand, it was abundantly expressed in fetal brain but not in fetal lung, liver or kidney tissues. The elevated level of transcript at immature stages and its subsequent decline In adult brain indicate that the encoded protein may be essential for development of the human brain.

Cloning, expression and mapping of a novel human zinc-finger gene TCF17 homologous to rodent Kid1

We isolated a novel human zinc-finger gene TCF17 homologous to rat Kid1, a zinc-finger gene of the Krüppel type expressed predominantly in kidney. In the rat this gene seems to be a transcription factor expressed in response to renal injury and ischemia. The 2435-bp human cDNA contained an open reading frame encoding 605 amino acids. The deduced amino acid sequence showed 86.0% and 87.2% identity (91.6% and 92.8% similarity) with the rat Kid1 and mouse Tcf17 respectively. In contrast with rat Kid1, human TCF17 was expressed in all human tissues examined, including kidney. This gene was mapped by FISH to chromosome 5q35.3, where cytogenetic and molecular abnormalities have been reported often in renal cell carcinomas.

Expression and chromosomal localization of KIAA0369, a putative kinase structurally related to Doublecortin

AbstractNeuropathy in vertebrates can be a consequence of failure of genes involved in the nervous system to be expressed at the correct times and levels during embryonic life. Recently, a brain specific gene, Doublecortin, was cloned and was shown to have mutations in X-linked lissencephaly and double cortex syndrome. KIAA0369 is a putative kinase that is structurally related to Doublecortin. We compared the expression of KIAA0369 with that of Doublecortin, both of which were expressed specifically or predominantly in fetal brain among 20 different tissues examined. The deduced products of both genes contain a unique domain (the Doublecortin [DC] domain), but KIAA0369 also contains a calmodulin-dependent kinase (CaM kinase)-like domain following the DC domain. We found at least four splicing variants of KIAA0369: KIAA0369-AS (type A, short version), KIAA0369-AL (type A, long version), KIAA0369-BS (type B, short version), and KIAA0369-BL (type B, long version). KIAA0369-B, which lacked the DC domain and maintained the kinase domain, was expressed in adult as well as fetal brain, but the variants that included the DC domain, KIAA0369-A, were expressed predominantly in fetal brain. These results suggest that the DC domain plays an important role in the development of the nervous system. In the adult brain, KIAA0369 was expressed in all 15 different regions examined, more intensely in cerebral cortex, occipital pole, frontal lobe, amygdala, and hippocampus, and less intensely in corpus callosum and thalamus. The murine homologs of Doublecortin and KIAA0369 were not detectable in 7-day mouse embryos, but both genes were expressed extensively in 11-day embryos. Human KIAA0369 was mapped by fluorescence in situ hybridization (FISH) to chromosome 13q13–q14.1. The presence of genes related to neuropathy has been reported in this locus.