Laminin-based cell adhesion anchors microtubule plus ends to the epithelial cell basal cortex through LL5α/β

LL5β has been identified as a microtubule-anchoring factor that attaches EB1/CLIP-associating protein (CLASP)–bound microtubule plus ends to the cell cortex. In this study, we show that LL5β and its homologue LL5α (LL5s) colocalize with autocrine laminin-5 and its receptors, integrins α3β1 and α6β4, at the basal side of fully polarized epithelial sheets. Depletion of both laminin receptor integrins abolishes the cortical localization of LL5s, whereas LL5 depletion reduces the amount of integrin α3 at the basal cell cortex. Activation of integrin α3 is sufficient to initiate LL5 accumulation at the cell cortex. LL5s form a complex with the cytoplasmic tails of these integrins, but their interaction might be indirect. Analysis of the three-dimensional distribution of microtubule growth by visualizing EB1-GFP in epithelial sheets in combination with RNA interference reveals that LL5s are required to maintain the density of growing microtubules selectively at the basal cortex. These findings reveal that signaling from laminin–integrin associations attaches microtubule plus ends to the epithelial basal cell cortex.     

A second generation of double mutant cholera toxin adjuvants: enhanced immunity without intracellular trafficking

Nasal application of native cholera toxin (nCT) as a mucosal adjuvant has potential toxicity for the CNS through binding to GM1 gangliosides in the olfactory nerves. Although mutants of cholera toxin (mCTs) have been developed that show mucosal adjuvant activity without toxicity, it still remains unclear whether these mCTs will induce CNS damage. To help overcome these concerns, in this study we created new double mutant CTs (dmCTs) that have two amino acid substitutions in the ADP-ribosyltransferase active center (E112K) and COOH-terminal KDEL (E112K/KDEV or E112K/KDGL). Confocal microscopic analysis showed that intracellular localization of dmCTs differed from that of mCTs and nCTs in intestinal epithelial T84 cells. Furthermore, both dmCTs exhibited very low toxicity in the Y1 cell assay and mouse ileal loop tests. When mucosal adjuvanticity was examined, both dmCTs induced enhanced OVA-specific immune responses in both mucosal and systemic lymphoid tissues. Interestingly, although both dmCT E112K/KDEV and dmCT E112K/KDGL showed high Th2-type and significant Th1-type cytokine responses by OVA-specific CD4+ T cells, dmCT E112K/KDEV exhibited significantly lower Th1-type cytokine responses than did nCT and dmCT E112K/KDGL. These results show that newly developed dmCTs retain strong biological adjuvant activity without CNS toxicity.     

p51/p63, a novel p53 homologue, potentiates p53 activity and is a human cancer gene therapy candidate

BACKGROUND: p51 (p73L/p63/p40/KET), a recently isolated novel p53 homologue, binds to p53-responsive elements to upregulate some p53 target genes and has been suggested to share partially overlapping functions with p53. p51 may be a promising candidate target molecule for anti-cancer therapy. METHODS: In this study, we adenovirally transduced p51A cDNA into human lung, gastric and pancreatic cancer cells and analyzed the intracellular function of p51 in anti-oncogenesis in vitro and in vivo. RESULTS: Overexpression of p51A revealed an anti-proliferative effect in vitro in all the cancer cells examined in this study. The anchorage-dependent and -independent cell growth of EBC1 cells carrying mutations in both p51 and p53 was suppressed and significant apoptosis following adenoviral transduction with p51 and/or p53 was seen. This growth suppression was cooperatively enhanced by the combined infection with adenoviral vectors encoding both p51 and p53. Furthermore, p51 activated several, but not all, p53-inducible genes, indicating that the mechanisms controlling p51- and p53-mediated tumor suppression differed. CONCLUSIONS: Our observations indicate that, although p51 exhibited reduced anti-oncogenetic effects compared with p53, it cooperatively enhanced the anti-tumor effects of p53. Our results suggest that p51 functions as a tumor suppressor in human cancer cells in vitro and in vivo and may be useful as a potential tool for cancer gene therapy.     

A novel mitochondrial ubiquitin ligase plays a critical role in mitochondrial dynamics

In this study, we have identified a novel mitochondrial ubiquitin ligase, designated MITOL, which is localized in the mitochondrial outer membrane. MITOL possesses a Plant Homeo-Domain (PHD) motif responsible for E3 ubiquitin ligase activity and predicted four-transmembrane domains. MITOL displayed a rapid degradation by autoubiquitination activity in a PHD-dependent manner. HeLa cells stably expressing a MITOL mutant lacking ubiquitin ligase activity or MITOL-deficient cells by small interfering RNA showed an aberrant mitochondrial morphology such as fragmentation, suggesting the enhancement of mitochondrial fission by MITOL dysfunction. Indeed, a dominant-negative expression of Drp1 mutant blocked mitochondrial fragmentation induced by MITOL depletion. We found that MITOL associated with and ubiquitinated mitochondrial fission protein hFis1 and Drp1. Pulse-chase experiment showed that MITOL overexpression increased turnover of these fission proteins. In addition, overexpression phenotype of hFis1 could be reverted by MITOL co-overexpression. Our finding indicates that MITOL plays a critical role in mitochondrial dynamics through the control of mitochondrial fission proteins.     

Fgf16 is essential for pectoral fin bud formation in zebrafish

Zebrafish pectoral fin bud formation is an excellent model for studying morphogenesis. Fibroblast growth factors (Fgfs) and sonic hedgehog (shh) are essential for pectoral fin bud formation. We found that Fgf16 was expressed in the apical ectodermal ridge (AER) of fin buds. A knockdown of Fgf16 function resulted in no fin bud outgrowth. Fgf16 is required for cell proliferation and differentiation in the mesenchyme and the AER of the fin buds, respectively. Fgf16 functions downstream of Fgf10, a mesenchymal factor, signaling to induce the expression of Fgf4 and Fgf8 in the AER. Fgf16 in the AER and shh in the zone of polarizing activity (ZPA) interact to induce and/or maintain each other's expression. These findings have revealed that Fgf16, a newly identified AER factor, plays a crucial role in pectoral fin bud outgrowth by mediating the interactions of AER-mesenchyme and AER-ZPA.     

Single-point mutations of hepatitis C virus NS3 that impair p53 interaction and anti-apoptotic activity of NS3

The N-terminal domain of NS3 of hepatitis C virus (HCV) possesses serine protease activity, which is essential for virus replication. This portion is also implicated in malignant transformation of hepatocytes. We previously demonstrated that an N-terminal portion of NS3 formed a complex with the tumor suppressor p53 and suppressed actinomycin D-induced apoptosis. We report here that single-point mutations of NS3 at position 106 from Leu to Ala (L106A), and position 43 from Phe to Ala (F43A) to a lesser extent, significantly impaired complex formation with p53. Moreover, the L106A mutation impaired an otherwise more distinct anti-apoptotic activity of NS3. F43A and L106A mutations also inhibited serine protease activity of NS3. These results collectively suggest the possibility that Leu106 and Phe43 are involved in p53 interaction and serine protease activity, and therefore, can be a good target for certain low-molecular-weight compound(s) to inhibit both oncogenic and replicative abilities of HCV.     

Geranylgeranyl-pyrophosphate (GGPP) synthase is down-regulated during differentiation of osteoblastic cell line MC3T3-E1

Isoprenylation of geranylgeranyl-pyrophosphate (GGPP) is critical for activation of small GTPases. We examined the roles of GGPP synthase (GGPPS) during the differentiation induced by the cell-to-cell contact in osteoblastic cell line MC3T3-E1 cells. We found that (1) both mRNA and protein expression of GGPPS was reduced with decrement of its activity during the differentiation, (2) GGOH, which is converted to GGPP in the cells, inhibited differentiation. These results suggest that the decrement of GGPP is critical for the cell-to-cell contact-induced differentiation, in which the down-regulation of GGPPS might be involved.     

Cytidine is a novel substrate for wild-type concentrative nucleoside transporter 2

Nucleoside transporter (NT) plays key roles in the physiology of nucleosides and the pharmacology of its analogues in mammals. We previously cloned Na+/nucleoside cotransporter CNT2 from mouse M5076 ovarian sarcoma cells, the peptide encoded by it differing from that by the previously reported mouse CNT2 in five substitutions, and observed that the transporter can take up cytidine, like CNT1 and CNT3. In the present study, we examined which of the two aforementioned CNT2 is the normal one, and whether or not cytidine is transported via the previously reported CNT2. The peptide encoded by CNT2 derived from mouse intestine, liver, spleen, and ovary was identical to that previously reported. The uptake of [3H]cytidine, but not [3H]thymidine, by Cos-7 cells transfected with CNT2 cDNA obtained from mouse intestine was much greater than that by mock cells, as in the case of [3H]uridine, a typical substrate of NT. [3H]Cytidine and [3H]uridine were taken up via CNT2, in temperature-, extracellular Na+-, and substrate concentration-dependent manners. The uptake of [3H]cytidine and [3H]uridine mediated by CNT2 was significantly inhibited by the variety of nucleosides used in this study, except for thymidine, and inhibition of the [3H]uridine uptake by cytidine was competitive. The [3H]uridine uptake via CNT2 was significantly decreased by the addition of cytarabin or gemcitabine, antimetabolites of cytidine analogue. These results indicated that the previously reported mouse CNT2 is the wild-type one, and cytidine is transported mediated by the same recognition site on the CNT2 with uridine, and furthermore, cytidine analogues may be substrates for the transporter.     

Sialidase NEU4 hydrolyzes polysialic acids of neural cell adhesion molecules and negatively regulates neurite formation by hippocampal neurons

Modulation of levels of polysialic acid (polySia), a sialic acid polymer, predominantly associated with the neural cell adhesion molecule (NCAM), influences neural functions, including synaptic plasticity, neurite growth, and cell migration. Biosynthesis of polySia depends on two polysialyltransferases ST8SiaII and ST8SiaIV in vertebrate. However, the enzyme involved in degradation of polySia in its physiological turnover remains uncertain. In the present study, we identified and characterized a murine sialidase NEU4 that catalytically degrades polySia. Murine NEU4, dominantly expressed in the brain, was found to efficiently hydrolyze oligoSia and polySia chains as substrates in sialidase in vitro assays, and also NCAM-Fc chimera as well as endogenous NCAM in tissue homogenates of postnatal mouse brain as assessed by immunoblotting with anti-polySia antibodies. Degradation of polySia by NEU4 was also evident in neuroblastoma Neuro2a cells that were co-transfected with Neu4 and ST8SiaIV genes. Furthermore, in mouse embryonic hippocampal primary neurons, the endogenously expressed NEU4 was found to decrease during the neuronal differentiation. Interestingly, GFP- or FLAG-tagged NEU4 was partially co-localized with polySia in neurites and significantly suppressed their outgrowth, whereas silencing of NEU4 showed the acceleration together with an increase in polySia expression. These results suggest that NEU4 is involved in regulation of neuronal function by polySia degradation in mammals.