Alzheimer's amyloid precursor protein (APP) 1 is an integral membrane protein with a receptor-like structure (1). A principal component of parenchymal amyloid deposits in Alzheimer's disease (AD) is ␤-amyloid (A␤) (2-4), which is derived from APP by proteolytic cleavage (1, 5-11). A␤ is thought to be generated through an intracellular protein secretory pathway of APP (for a review, see Ref. 12). The short APP cytoplasmic domain consisting of 47 amino acid residues is thought to be responsible for determination of APP metabolism (13-15) and possible signal transduction from a putative extracellular ligand that has yet to be identified (for a review, see Ref. 16). Because APP is a candidate pathogenic factor of AD, elucidation of the physiological function of APP as well as the determination of the metabolic mechanism of A␤ production should increase our understanding of the pathogenesis of AD. Using a yeast two-hybrid system, we isolated cDNA of proteins that interact with the cytoplasmic domain of APP (APP COOH ) in order to elucidate the molecular mechanisms of APP metabolism and function of APP. Previous efforts to identify and isolate proteins associating with APP COOH , utilizing the yeast two-hybrid system, have resulted in the isolation of proteins carrying phosphotyrosine binding/phosphotyrosine interaction (PI) domains such as Fe65 (17), 19), and X11 (20). The X11 gene, which is located on chromosome 9, was originally isolated as a gene candidate for Friedreich ataxia (21) and its partial cDNA was identified as a clone encoding a protein that associates with APP COOH (22). The PI domain of X11 interacts with the YENPTY motif of APP COOH (20). In the present study, we isolated a complete cDNA encoding an X11-like protein (X11L) from a human adult brain cDNA library, utilizing the yeast two-hybrid system and APP COOH as a bait. A partial short cDNA encoding approximately 190 amino acids in the PI domain of X11L has already been isolated using a similar procedure (22). However, detailed characterization of X11L binding to APP and identification of the role X11L plays in the physiological function of APP have not been performed. We found that human X11L requires a sequence containing the NPXY motif of APP COOH for APP binding and that association of the PI domain with APP was suppressed by a deletion of a amino-terminal domain fused to the PI domain (PI ϩ C construct) but enhanced by a deletion of a carboxyl-terminal domain fused to the PI domain (N ϩ PI construct). Co-transfection of full-length human X11L into cells that express stably transfected human APP695 cDNA resulted in decreased secretion of A␤40 but not A␤42. However, co-transfection into cells of the cDNA lacking the carboxyl-terminal domain (N ϩ PI construct) or a cDNA encoding only the PI domain (PI construct), whose protein products preserve the ability to bind to APP COOH , did not present the ability to modulate A␤ production. The present results suggest that the amino-terminal region of the PI domain is needed to regulate binding affinity...

Estrogen plays an important role in many physiological events including carcinogenesis and the development of human breast cancer. However, the molecular mechanisms of estrogen signaling in cancers have not been clarified hitherto and accurate therapeutic prediction of breast cancer is earnestly desired. We first carried out estrogen-responsive expression profiling of approximately 9000 genes in estrogen receptor-positive human MCF-7 breast cancer cells. Based on the results, estrogen-responsive genes were selected for production of a custom-made cDNA microarray. Using a microarray consisting of the narrowed-down gene subset, we first analyzed the time course of the estrogen-responsive gene expression profiles in MCF-7 cells, resulting in subdivision of the genes up-regulated by estrogen into early-responsive and late-responsive genes. The expression patterns of several genes were confirmed by Northern blot analysis. We also analyzed the effects of the estrogen antagonists ICI 182,780 and 4-hydroxytamoxifen (OHT) on the estrogen-responsive gene expression profiles in MCF-7 cells. While the regulation of most of the genes by estrogen was completely abolished by ICI 182,780, some genes were partially regulated by estrogen even in the presence of OHT. Furthermore, the estrogen-responsive gene expression profiles of twelve cancer cell lines derived from the breast, ovary, stomach and other tissues were obtained and analyzed by hierarchical clustering including the profiles in MCF-7 cells. Several genes also showed up-regulation or down-regulation by estrogen in cell lines other than MCF-7 cells. The significance of the estrogen-responsive genes identified in these analyses concerning the nature of cancer is discussed.

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Calmodulin-dependent nitric-oxide synthase, with an apparent molecular mass of 125 kDa, was induced in the liver of rats treated with Propionibacterium acnes and Escherichiu coli lipopolysaccharide. Clones were isolated from a cDNA library obtained from induced rat liver using oligonucleotide probes which were synthesized based on the amino acid sequences of peptides of the purified enzyme. Four overlapping cDNA clones for a 3.8-kbp region were isolated and the nucleotide sequences were determined. These clones encompassed an open-reading frame of 3441 bases encoding 1147 amino acids. The deduced amino acid sequence of the cDNA suggested that thc protein contains binding sites for NADPH, FAD and FMN. The structure of the possible calmodulinbinding site, consisting of a strongly hydrophobic region surrounded by basic amino acids, is present. The full-length cDNA was expressed in COS 1 cells under the control of a cytomegalovirus promoter and the expressed enzyme was found to be a calmodulin-dependent nitric-oxide synthase. A structural comparison suggested that the liver nitric-oxide synthase is the same as the macrophage enzyme. Northern-blot analysis showed that the mRNA in the liver is approximately 4.2 kb long and is induced transcriptionally by treatment with P acnes and lipopolysaccharide.Many biological roles are attributed to a newly identified small molecule, nitric oxide, which is generated from L-arginine by nitric-oxide (NO) synthase [l, 21. It is of interest to determine whether the molecular diversity of NO synthase indicates functional diversity for the enzyme. The molecular diversity of NO synthase is not yet fully understood, but at least two major forms are recognized; the constitutive and inducible Caz+/calmodulin-dependent form and the Ca"/ calmodulin-independent form [ 11.The nucleotide sequence of rat brain Ca2 ' kalmodulindependent constitutive NO synthase has been determined [ 3 ] . In this study, it was found that brain NO synthase contains the calmodulin-binding site as well as binding sites for FAD, FMN and NADPH. More recently, other studies suggest that NO synthases also contain heme and putative heme binding sites [4, 51. It was observed that among the isozymes of neuronal NO synthase and endothelial NO synthase these binding sites are well conserved 161. Information on the structure of inducible NO synthase is also accumulating. However, regarding the inducible NO synthase, there is an obvious discrepancy in studies relating to the calmodulin dependence of the enzyme [7-111. Inducible NO synthases from macro- Note. The novel nucleotide sequence data published here have been submitted to the EMBL/GenBank sequence data bank(s) and are available under accession number(s) D12520. phages and polymorphonuclear leukocytes have been purified and characterized. These inducible forms of NO synthases are reported to be Ca'+/calmodulin independent, in contrast to the constitutive form [7,11, 121. This calmodulin independence was confirmed by the molecular cloning and sequence determination of m...

Alzheimer amyloid precursor protein (APP) is an integral membrane protein with a short cytoplasmic domain of 47 amino acids. It is hoped that identification of proteins that interact with the cytoplasmic domain will provide new insights into the physiological function of APP and, in turn, into the pathogenesis of Alzheimer's disease. To identify proteins that interact with the cytoplasmic domain of APP, we employed affinity chromatography using an immobilized synthetic peptide corresponding to residues 645-694 of APP 695 and identified a protein of ϳ130 kDa in rat brain cytosol. Amino acid sequencing of the protein revealed the protein to be a rat homologue of monkey UV-DDB (UV-damaged DNAbinding protein, calculated molecular mass of 127 kDa). UV-DDB/p127 co-immunoprecipitated with APP using an anti-APP antibody from PC12 cell lysates. APP also coimmunoprecipitated with UV-DDB/p127 using an anti-UV-DDB/p127 antibody. These results indicate that UV-DDB/p127, which is present in the cytosolic fraction, forms a complex with APP through its cytoplasmic domain. In vitro binding experiments using a glutathione S-transferase -APP cytoplasmic domain fusion protein and several mutants indicated that the YENPTY motif within the APP cytoplasmic domain, which is important in the internalization of APP and amyloid ␤ protein secretion, may be involved in the interaction between UV-DDB/ p127 and APP. Key Words: Amyloid precursor protein-UV-damaged DNA binding protein-DNA repair.