There is no proof that the HN cDNA represents a gene, that its origin is nuclear, or that the HN peptide is produced in vivo. The information that the long HN cDNA sequence is virtually identical to mitochondrial rRNA should have been put in the Discussion rather than published as supplementary material. The Discussion should have contained the following:The long cDNA [1,567 bp including a poly(A) tail] containing the HN ORF is Ͼ99% identical (1548͞1552) to positions 1679-3230 of mitochondrial DNA (GenBank accession no. AB055387). Because mitochondrial DNA positions 1667-3224 code for mitochondrial 16S rRNA, and mitochondrial 16S rRNA has a short poly(A) tail during transcription (1), the virtual identity of the long HN cDNA to mitochondrial DNA indicates that HN cDNA is mitochondrial 16S rRNA with a poly(A) tail. This makes it unlikely that the peptide encoded by the ORF in HN cDNA is naturally produced. Further, the 75-bp HN ORF is separated from the 5Ј end of the long HN cDNA by a 950-bp region containing at least seven ORFs, each with a stop codon. This makes it even more unlikely that HN peptide is produced from the long HN cDNA. It should also be noted that mitochondria-like nuclear sequences occur commonly as pseudogenes (2). Finally, the HN peptide lacks the characteristic N-terminal signal sequence of secreted peptides, although we suggest that a signal peptide-like function may be encoded in the primary sequence of HN peptide. For all of these reasons, it is unlikely that the HN ORF leads to production of the predicted peptide in vivo.Nonetheless, it remains possible that HN cDNA represents a nuclear transcribed mRNA and that the HN peptide is a natural product. Long regions of the HN cDNA are Ͼ99% identical to certain registered human mRNAs [1545͞1553 at positions 14-1580 of FLJ22981 fis cDNA (AK026634), 925͞929 at positions 1-929 of FLJ22517 fis cDNA, 914͞919 at positions 1348-2266 of FLJ20341 fis cDNA, and 345͞346 at positions 1-346 of PNAS-32 mRNA]. PNAS-32 mRNA is actually expressed to produce NB4 apoptosis-related protein, showing that this mRNA is transcribed from a nuclear gene. In addition, HN cDNA is highly similar to regions of more than 1,000 bp on human chromosomes [positions 245364-244075 of chromosome 11 draft sequence (92%, 1198͞1290), positions 65752-66775 of chromosome X draft sequence (95%, 974͞1025), and positions 687598-688608 of chromosome 5 draft sequence (93%, 954͞1016)]. Also, the HN ORF has a Kozak-like sequence, although it is not canonical.

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A novel factor, termed Humanin (HN), antagonizes against neurotoxicity by various types of familial Alzheimer's disease (AD) genes [V642I and K595N/M596L (NL) mutants of amyloid precursor protein (APP), M146L-presenilin (PS) 1, and N141I-PS2] and by A␤1-43 with clear action specificity ineffective on neurotoxicity by polyglutamine repeat Q79 or superoxide dismutase 1 mutants. Here we report that HN can also inhibit neurotoxicity by other AD-relevant insults: other familial AD genes (A617G-APP, L648P-APP, A246E-PS1, L286V-PS1, C410Y-PS1, and H163R-PS1), APP stimulation by anti-APP antibody, and other A␤ peptides (A␤1-42 and A␤25-35). The action specificity was further indicated by the finding that HN could not suppress neurotoxicity by glutamate or prion fragment. Against the AD-relevant insults, essential roles of Cys 8 and Ser 14 were commonly indicated, and the domain from Pro 3 to Pro 19 was responsible for the rescue action of HN, in which seven residues turned out to be essential. We also compared the neuroprotective action of S14G HN (HNG) with that of activity-dependent neurotrophic factor, IGF-I, or basic FGF for the antagonism against various AD-relevant insults (V642I-APP, NL-APP, M146L-PS1, N141I-PS2, and A␤1-43). Although all of these factors could abolish neurotoxicity by A␤1-43, only HNG could abolish cytotoxicities by all of them. HN and HN derivative peptides may provide a new insight into the study of AD pathophysiology and allow new avenues for the development of therapeutic interventions for various forms of AD.

Surface tension measurements were carried out at 20 degrees C by a capillary drop-weight method on aqueous solutions of sodium glutamate (NaGlu), lysine hydrochloride (LysHCl), potassium aspartate (KAsp), arginine hydrochloride (ArgHCl), lysylglutamate (LysGlu), argininylglutamate (ArgGlu), guanidinium sulfate, trehalose, trimethylamine N-oxide (TMAO), dimethyl sulfoxide, 2-methyl-2,4-pentanediol (hexylene glycol), and poly(ethylene glycol)s of molecular weights 200, 400, 600, and 1000. All of the salts and the sugar increased the surface tension of water, while the last four compounds decreased it, with 2-methyl-2,4-pentanediol lowering it most effectively and TMAO being the least effective. The preferential hydration of bovine serum albumin (BSA) and lysozyme was measured in KAsp, ArgHCl, LysGlu, and ArgGlu. The high values of preferential hydration found in all cases, except for BSA in ArgHCl, suggest that they should stabilize protein structure, as had been found for lysine hydrochloride and monosodium glutamate [Arakawa, T., & Timasheff, S. N. (1984) J. Biol. Chem. 259, 4979-4986]. A correlation was found for both BSA and lysozyme in KAsp, NaGlu, LysHCl, ArgGlu, and LysGlu between the surface tension effect and the observed preferential interactions, indicating that the change in the surface free energy of the protein-containing cavity due to the surface tension increase for water by these amino acid salts contributes dominantly to the observed increase in the chemical potential of the protein by their addition. The lack of a correlation observed for BSA, but not lysozyme, in ArgHCl at low concentrations where preferential binding is close to zero suggests, however, that the surface tension effect is not the sole factor involved in the protein-solvent interactions in these amino acid salts. Binding of ArgHCl to BSA, probably through hydrogen bonds between the Arg guanidinium group and peptide bonds, was proposed to occur, the affinity of Arg+ being reduced by electrostatic repulsion when proteins carry a net positive charge, such as is the case with lysozyme. Since the four organic solvent additives also lead to protein preferential hydration, no correlation exists between their preferential interactions and the surface free energy perturbation. Therefore, in their case, the preferential hydration must be ascribed to other factors that overcome the preferential binding expected from the Gibbs adsorption isotherm. The surface tension results, however, are consistent with the binding of the organic solvents to proteins through hydrophobic interactions, explaining, at least in part, the observed concentration dependence of the interactions.

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Using a yeast two-hybrid method, we searched for amyloid precursor protein (APP)-interacting molecules by screening mouse and human brain libraries. In addition to known interacting proteins containing a phosphotyrosine-interaction-domain (PID)-Fe65, Fe65L, Fe65L2, X11, and mDab1, we identified, as a novel APP-interacting molecule, a PID-containing isoform of mouse JNK-interacting protein-1 (JIP-1b) and its human homolog IB1, the established scaffold proteins for JNK. The APP amino acids Tyr(682), Asn(684), and Tyr(687) in the G(681)YENPTY(687) region were all essential for APP/JIP-1b interaction, but neither Tyr(653) nor Thr(668) was necessary. APP-interacting ability was specific for this additional isoform containing PID and was shared by both human and mouse homologs. JIP-1b expressed by mammalian cells was efficiently precipitated by the cytoplasmic domain of APP in the extreme Gly(681)-Asn(695) domain-dependent manner. Reciprocally, both full-length wild-type and familial Alzheimer's disease mutant APPs were precipitated by PID-containing JIP constructs. Antibodies raised against the N and C termini of JIP-1b coprecipitated JIP-1b and wild-type or mutant APP in non-neuronal and neuronal cells. Moreover, human JNK1beta1 formed a complex with APP in a JIP-1b-dependent manner. Confocal microscopic examination demonstrated that APP and JIP-1b share similar subcellular localization in transfected cells. These data indicate that JIP-1b/IB1 scaffolds APP with JNK, providing a novel insight into the role of the JNK scaffold protein as an interface of APP with intracellular functional molecules.

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