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.

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.

Background Merkel cell carcinoma (MCC) is an aggressive skin cancer with a recurrence rate of >40%. Of the 2000 MCC cases/year in the USA, most are caused by the Merkel cell polyomavirus (MCPyV). Antibodies to MCPyV-oncoprotein (T-antigens) have been correlated with MCC tumor burden. We prospectively validated the clinical utility of MCPyV oncoprotein antibody titers for MCC prognostication and surveillance. Methods MCPyV-oncoprotein antibody detection was optimized in a clinical laboratory. A cohort of 219 patients with newly-diagnosed MCC were followed prospectively (median follow-up 1.9 years). Among seropositive patients, antibody titer and disease status were serially tracked. Results Antibodies to MCPyV-oncoproteins were rare among healthy individuals (1%) but present in most MCC patients (114 of 219, 52%, p<0.01). Seropositivity at diagnosis independently predicted decreased recurrence risk (HR=0.58; p=0.04) in multivariate analyses adjusted for age, sex, stage, and immunosuppression. Following initial treatment, seropositive patients whose disease did not recur had rapidly falling titers that became negative by a median of 8.4 months. Among seropositive patients who underwent serial evaluation (71 patients; 282 timepoints), an increasing oncoprotein titer had a positive predictive value of 66% for clinically evident recurrence while a decreasing titer had a negative predictive value of 97%. Conclusions Determination of oncoprotein antibody titer assists in the clinical management of newly diagnosed MCC patients by stratifying them into a higher risk seronegative cohort in whom radiologic imaging may play a more prominent role, and into a lower-risk seropositive cohort whose disease status can be tracked in part via oncoprotein antibody titer.

In the yeast Saccharomyces cerevisiae, Sic1, an inhibitor of Clb-Cdc28 kinases, must be phosphorylated and degraded in G1 for cells to initiate DNA replication, and Cln-Cdc28 kinase appears to be primarily responsible for phosphorylation of Sic1. The Pho85 kinase is a yeast cyclin-dependent kinase (Cdk), which is not essential for cell growth unless both CLN1 and CLN2 are absent. We demonstrate that Pho85, when complexed with Pcl1, a G1 cyclin homologue, can phosphorylate Sic1 in vitro, and that Sic1 appears to be more stable in pho85Delta cells. Three consensus Cdk phosphorylation sites present in Sic1 are phosphorylated in vivo, and two of them are required for prompt degradation of the inhibitor. Pho85 and other G1 Cdks appear to phosphorylate Sic1 at different sites in vivo. Thus at least two distinct Cdks can participate in phosphorylation of Sic1 and may therefore regulate progression through G1.

Amyloid precursor protein (APP), the precursor of Ab, has been shown to function as a cell surface receptor that mediates neuronal cell death by anti-APP antibody. The c-Jun N-terminal kinase (JNK) can mediate various neurotoxic signals, including Ab neurotoxicity. However, the relationship of APP-mediated neurotoxicity to JNK is not clear, partly because APP cytotoxicity is Ab independent. Here we examined whether JNK is involved in APP-mediated neuronal cell death and found that: (i) neuronal cell death by antibody-bound APP was inhibited by dominant-negative JNK, JIP-1b and SP600125, the specific inhibitor of JNK, but not by SB203580 or PD98059; (ii) constitutively active (ca) JNK caused neuronal cell death and (iii) the pharmacological profile of caJNK-mediated cell death closely coincided with that of APP-mediated cell death. Pertussis toxin (PTX) suppressed APP-mediated cell death but not caJNK-induced cell death, which was suppressed by Humanin, a newly identified neuroprotective factor which inhibits APP-mediated cytotoxicity. In the presence of PTX, the PTX-resistant mutant of Ga o , but not that of Ga i , recovered the cytotoxic action of APP. These findings demonstrate that JNK is involved in APP-mediated neuronal cell death as a downstream signal transducer of G o .

The 24-residue peptide Humanin (HN), containing two Ser residues at positions 7 and 14, protects neuronal cells from insults of various Alzheimer's disease (AD) genes and Ab. It was not known why the rescue function of (S14G)HN is more potent than HN by two to three orders of magnitude. Investigating the possibility that the post-translational modification of Ser14 might play a role, we found that HN with D-Ser at position 14 exerts neuroprotection more potently than HN by two to three orders of magnitude, whereas D-Ser7 substitution does not affect the rescue function of HN. On the other hand, S7A substitution nullified the HN function. Multiple series of experiments indicated that Ser7 is necessary for self-dimerization of HN, which is essential for neuroprotection by this factor. These findings indicate that the rescue function of HN is quantitatively modulated by D-isomerization of Ser14 and Ser7-relevant dimerization, allowing for the construction of a very potent HN derivative that was fully neuroprotective at 10 pM against 25 lM Ab1-43. This study provides important clues to the understanding of the neuroprotective mechanism of HN, as well as to the development of novel AD therapeutics. Keywords: Alzheimer's disease, dimerization, Humanin, neuronal death, neuroprotection, D-serine isomerization. Alzheimer's disease (AD) is the most prevalent neurodegenerative disease associated with progressive dementia. No fundamental therapy for this disease has so far been established. As brain atrophy is the central abnormality in AD, pathological mechanisms leading to neuronal loss must be understood to develop future curative therapy. Three forms of known mutant genes cause familial AD (FAD): amyloid precursor protein (APP) mutants, presenilin (PS)1 mutants, and PS2 mutants (Shastry and Giblin 1999). Multiple groups have found that all examined FAD mutants cause or enhance cell death when they are expressed in neuronal cells (Niikura et al. 2002 for review). In an attempt to find the molecules that suppress neuronal cell death by these AD-related insults, we used 'death-trap' screening, developed by D'Adamio et al. (1997) -an unbiased functional screening of molecules that allow dying cells to survive.We applied this method to V642I-APP-inducible neuronal cells with our unique modification using an expression cDNA library constructed from an occipital lobe of the brain of an autopsy-diagnosed AD patient. As a result of this screening, we identified an ORF cDNA, encoding a novel short peptide MAP-RGFSCLLLLTSEIDLPVKRRA, that suppresses neuronal cell death by various FAD genes [APP mutants (V642I, K595M/N596L, A617G, and L648P), PS1 mutants (M146L, H163R, A246E, L286V, and C410Y), and a PS2 mutant Received February 20, 2003; accepted March 7, 2003. Address correspondence and reprint requests to Ikuo Nishimoto, Departments of Pharmacology and Anatomy, KEIO University School of Medicine, Medical Research Center, 6th Floor, 35 Shinanomachi, Tokyo 160-8582, Japan. E-mail: nisimoto@sc.itc.keio.ac.jp 1 These two ...

Humanin (HN) is a newly identified neuroprotective peptide that specifically suppresses Alzheimer's disease (AD)-related neurotoxicity. HN peptide has been detected in the human AD brain as well as in mouse testis and colon by immunoblot and immunohistochemical analyses. By means of yeast two-hybrid screening, we identified TRIM11 as a novel HN-interacting protein. TRIM11, which is a member of protein family containing a tripartite motif (TRIM), is composed of a RING finger domain, which is a putative E3 ubiquitin ligase, a B-box domain, a coiled-coil domain and a B30.2 domain. Deletion of the B30.2 domain in TRIM11 abolished the interaction with HN, whereas the B30.2 domain alone did not interact with HN. For their interaction, at least the coiled-coil domain was indispensable together with the B30.2 domain. The intracellular level of glutathione S-transferase-fused or EGFP-fused HN peptides or plain HN was drastically reduced by the coexpression of TRIM11. Disruption of the RING finger domain by deleting the first consensus cysteine or proteasome inhibitor treatment significantly diminished the effect of TRIM11 on the intracellular level of HN. These results suggest that TRIM11 plays a role in the regulation of intracellular HN level through ubiquitin-mediated protein degradation pathways.

Novel approaches to prevent and reverse ultraviolet (UV) damage are needed to combat rising sunlight-induced skin cancer rates. Mouse studies have shown that oral or topical caffeine promotes elimination of UV-damaged keratinocytes via apoptosis and markedly inhibits subsequent skin cancer development. This potentially important therapeutic effect has not been studied in human skin cells. Here we use primary human keratinocytes to examine which of several caffeine effects mediates this process. In these cells, caffeine more than doubled apoptosis after 75 mJ/cm2 of UVB. Selectively targeting two of caffeine’s known effects did not alter UVB-induced apoptosis: inhibition of ATM (ataxia-telangiectasia mutated) and augmentation of cyclic AMP levels. In contrast, siRNA against ATR (ataxia-telangiectasia and Rad3-related) doubled apoptosis after UV through a p53-independent mechanism. Caffeine did not further augment apoptosis after UVB in cells in which ATR had been specifically depleted, suggesting a key target of caffeine in this effect is ATR. Inhibition of a central ATR target, Chk1, via siRNA or a novel highly specific inhibitor (PF610666) also augmented UVB-induced apoptosis. These data suggest that a relevant target of caffeine is the ATR-Chk1 pathway and that inhibiting ATR or Chk1 may have promise in preventing or reversing UV damage.