Structural basis of sugar-recognizing ubiquitin ligase

SCF(Fbs1) is a ubiquitin ligase that functions in the endoplasmic reticulum (ER)-associated degradation pathway. Fbs1/Fbx2, a member of the F-box proteins, recognizes high-mannose oligosaccharides. Efficient binding to an N-glycan requires di-N-acetylchitobiose (chitobiose). Here we report the crystal structures of the sugar-binding domain (SBD) of Fbs1 alone and in complex with chitobiose. The SBD is composed of a ten-stranded antiparallel beta-sandwich. The structure of the SBD-chitobiose complex includes hydrogen bonds between Fbs1 and chitobiose and insertion of the methyl group of chitobiose into a small hydrophobic pocket of Fbs1. Moreover, NMR spectroscopy has demonstrated that the amino acid residues adjoining the chitobiose-binding site interact with the outer branches of the carbohydrate moiety. Considering that the innermost chitobiose moieties in N-glycans are usually involved in intramolecular interactions with the polypeptide moieties, we propose that Fbs1 interacts with the chitobiose in unfolded N-glycoprotein, pointing the protein moiety toward E2 for ubiquitination.     

Acoustic stiffness and change in plug cartilage over time after autologous osteochondral grafting: correlation between ultrasound signal intensity and histological score in a rabbit model

We investigated quantitative changes over time in ultrasound signal intensity (an index of stiffness), signal duration (an index of surface irregularity), and interval between signals (an index of thickness) of plug cartilage in an animal model of autologous osteochondral grafting. A full-thickness osteochondral plug was surgically removed and replaced in male Japanese white rabbits (n = 22). Specimens obtained at day 0 and weeks 2, 4, 8, 12 and 24 postoperatively were assessed using an ultrasound system and by macroscopic and histological evaluation (modified Mankin's score). Histology revealed that the plug sank until 2 weeks postoperatively, and that newly formed cartilage-like tissue covered the plug, but at 24 weeks the tissue detached. The plug itself survived well throughout the period of observation. Although the signal intensity at the plug site was same as that in the sham operated contralateral knee at day 0, from 2 to 24 weeks postoperatively it was less than that in the sham knee. At 8 weeks, this difference was significant (P < 0.05). Modified Mankin's score revealed early degenerative changes at the site, but macroscopic examination did not. Signal intensity correlated significantly with score (both at day 0 and at the five postoperative time points [P < 0.05, r = -0.91] and as a whole [P < 0.05, r = -0.36]). Signal intensity also significantly correlated with the individual subscores for 'cartilage structure' (P < 0.05, r = -0.32) and 'cartilage cells' (P < 0.05, r = -0.30) from the modified Mankin's score, but not significantly with subscores for 'staining' and 'tidemark'. Signal duration correlated significantly with total score (as a whole [P < 0.05, r = 0.34]), but not significantly with the score for cartilage structure (P = 0.0557, r = 0.29). The interval between signals reflected well the actual thickness of the plug site. The significant relationships between ultrasound signal intensity and scores suggest that early degenerative changes in plug cartilage and cartilage-like tissue, especially in the superficial layer, are detectable by high-frequency ultrasound assessment.     

(3Z)-2-Acetylamino-3-octadecen-1-ol as a potent apoptotic agent against HL-60 cells

(2R,3Z)-, (2R,3E)-, (2S,3Z) and (2S,3E)-2-Acetylamino-3-octadecen-1-ol, and (2R)- and (2S)-2-acetylamino-octadecan-1-ol were prepared using the Wittig olefination of Garner's aldehyde (N-Boc-N,O-isopropylidene-L- or D-serinal) from L- or D-serine. The apoptotic activities of these saturated and unsaturated 2-acetylaminoalcohols were examined in human leukemia HL-60 cells using MTT assay. Among the newly synthesized compounds, the cis-isomers were the most potent. Despite their simple structures, (2R,3Z)- and (2S,3Z)-2-acetylamino-3-octadecen-1-ol showed high and comparable apoptotic activities compared with N-acetyl-D-erythro-sphingosine (D-e-C2-Cer, a well-known inducer of apoptosis). Their apoptotic activities were in the order D-e-C2-Cer approximately L-e-C2-Cer approximately (2R,3Z)- approximately (2S,3Z)->(2R,3E)- approximately (2S,3E)- approximately (2R)- approximately (2S)-derivative. Qualitative analysis of DNA fragmentation caused by these compounds was conducted using agarose gel electrophoresis, and typical DNA fragmentation was found in the cases of (2R,3Z)- and (2S,3Z)-isomers such as C2-Cer, but not trans and saturated isomers. The morphological features of the cells, the proteolytic processing of pro-caspase-3, and the cleavage of PARP as a result of exogenous treatment with (2R,3Z)- and (2S,3Z)-isomers indicated that cell death induced by these compounds was apoptosis. These observations suggest that these newly synthesized compounds, (3Z)-2-Acetylamino-3-octadecen-1-ol, have similar characteristics and apoptosis-inducing activities against HL-60 cells with C2-Cer.     

Cyclic ADP-ribose, a putative Ca2+-mobilizing second messenger, operates in submucosal gland acinar cells

Cyclic ADP-ribose (cADPR), a putative Ca(2+)-mobilizing second messenger, has been reported to operate in several mammalian cells. To investigate whether cADPR is involved in electrolyte secretion from airway glands, we used a patch-clamp technique, the measurement of microsomal Ca(2+) release, quantification of cellular cADPR, and RT-PCR for CD38 mRNA in human and feline tracheal glands. cADPR (>6 microM), infused into the cell via the patch pipette, caused ionic currents dependent on cellular Ca(2+). Infusions of lower concentrations (2-4 microM) of cADPR or inositol 1,4,5-trisphosphate (IP(3)) alone were without effect on the baseline current, but a combined application of cADPR and IP(3) mimicked the cellular response to low concentrations of acetylcholine (ACh). Microsomes derived from the isolated glands released Ca(2+) in response to both IP(3) and cADPR. cADPR released Ca(2+) from microsomes desensitized to IP(3) or those treated with heparin. The mRNA for CD38, an enzyme protein involved in cADPR metabolism, was detected in human tissues, including tracheal glands, and the cellular content of cADPR was increased with physiologically relevant concentrations of ACh. We conclude that cADPR, in concert with IP(3), operates in airway gland acinar cells to mobilize Ca(2+), resulting in Cl(-) secretion.     

Synthesis of non-natural C2-homo-ceramide and its apoptotic activity against HL-60 cells

Non-natural ceramide analogues, C2-homo-ceramide and C2-homo-dihydroceramide, were prepared from L-aspartic acid via L-homo-serine. The apoptotic activities of the synthesized ceramide analogues were examined in HL-60 human leukemia cells. C2-homo- and C2-bishomo-ceramide indicate low but considerable apoptotic activities in comparison with C2-ceramide.     

Patch establishment and development of a clonal plant,Polygonum cuspidatum,on Mount Fuji

Microsatellite analysis was used to investigate the patch establishment and development of Polygonum cuspidatum Sieb. et Zucc, a clonal herbaceous plant that dominates the primary succession on the southeast slope of Mount Fuji. Genotypes of P. cuspidatum in 155 patches at the study site differed from each other. This indicates that P. cuspidatum patches are initially established by seed dispersed on the bare scoria field, and not by clonal rhizome extension. Genetic differentiation was estimated using the FST values between subpopulations at the study site. There was almost no genetic differentiation between subpopulations, indicating the presence of massive gene flow. The pollen fathers of seeds and maternal genets of current-year seedlings were inferred from the microsatellite allele composition by a simple exclusion method. The wide, random distribution of pollen fathers suggests that pollen dispersal occurs over a broad area. Maternal analysis showed a tendency for seed dispersal to be biased to the area nearby and down slope from the mother plants. Patch establishment under massive gene flow may result from such pollen and seed dispersal. To understand the process of patch development, aerial photographs taken from 1962 to 1999 were compared, and then genets in each of 36 patches were identified from the microsatellite genotypes of P. cuspidatum shoots. The comparison of aerial photographs showed that most of the patches enlarged each year and that some neighbouring patches combined during growth. Genet analysis demonstrated a high correlation between patch area and the area of the largest genet within it, and that new genets were recruited at the patch periphery. These findings indicate that both vegetative and sexual reproduction, i.e. rhizome extension and the establishment of new seedlings, contribute to the development of P. cuspidatum patches.     

Genetic structure and reproduction dynamics of Salix reinii during primary succession on Mount Fuji,as revealed by nuclear and chloroplast microsatellite analysis

The early stage of volcanic desert succession is underway on the southeastern slope of Mount Fuji. We used markers of nuclear microsatellites (simple sequence repeats; SSR) and chloroplast microsatellites (cpSSR) to investigate the population genetic structure and reproduction dynamics of Salix reinii, one of the dominant pioneer shrubs in this area. The number of S. reinii genets in a patch and the area of the largest genet within the patch increased with patch area, suggesting that both clonal growth and seedling recruitment are involved in the reproduction dynamics of S. reinii. Five polymorphic cpSSR markers were developed for S. reinii by sequencing the noncoding regions between universal sequences in the chloroplast genome. Nineteen different cpSSR haplotypes were identified, indicating that S. reinii pioneer genets were created by the long-distance dispersal of seeds originating from different mother genets around the study site, where all vegetation was destroyed during the last eruption. Furthermore, the clustered distributions of different haplotypes within each patch or plot suggested that newly colonized genets tended to be generated from seeds dispersed near the initially established mother genets. These results revealed that the establishment of the S. reinii population on the southeastern slope of Mount Fuji involved two sequential modes of seed dispersal: long-distance dispersal followed by short-distance dispersal.     

Apoptotic activities of C2-ceramide and C2-dihydroceramide homologues against HL-60 cells

The apoptotic activities of non-natural ceramide homologues, C2-homo-ceramide, C2-homo-dihydroceramide, C2-bishomo-ceramide and C2-bishomo-dihydroceramide, were examined using human leukemia HL-60 cells. The apoptotic activity was in order of C2-ceramide>C2-homo-ceramide approximately C2-bishomo-ceramide and the activities of the L-erythro- and D-erythro-ceramide homologues were similar. The morphological features of the cells, DNA fragmentations, proteolytic processing of pro-caspase-3 and the cleavage of PARP as the result of treatments with these homologues indicated that cell death was induced by apoptosis.     

A palmitoylated RING finger ubiquitin ligase and its homologue in the brain membranes

Ubiquitin (Ub) ligation is implicated in active protein metabolism and subcellular trafficking and its impairment is involved in various neurologic diseases. In rat brain, we identified two novel Ub ligases, Momo and Sakura, carrying double zinc finger motif and RING finger domain. Momo expression is enriched in the brain gray matter and testis, and Sakura expression is more widely detected in the brain white matter as well as in many peripheral organs. Both proteins associate with the cell membranes of neuronal and/or glial cells. We examined their Ub ligase activity in vivo and in vitro using viral expression vectors carrying myc-tagged Momo and Sakura. Overexpression of either Momo or Sakura in mixed cortical cultures increased total polyubiquitination levels. In vitro ubiquitination assay revealed that the combination of Momo and UbcH4 and H5c, or of Sakura and UbcH4, H5c and H6 is required for the reaction. Deletion mutagenesis suggested that the E3 Ub ligase activity of Momo and Sakura depended on their C-terminal domains containing RING finger structure, while their N-terminal domains influenced their membrane association. In agreement, Sakura associating with the membrane was specifically palmitoylated. Although the molecular targets of their Ub ligation remain to be identified, these findings imply a novel function of the palmitoylated E3 Ub ligase(s).     

Proteasome activator PA28gamma-dependent nuclear retention and degradation of hepatitis C virus core protein

Hepatitis C virus (HCV) core protein plays an important role in the formation of the viral nucleocapsid and a regulatory protein involved in hepatocarcinogenesis. In this study, we have identified proteasome activator PA28gamma (11S regulator gamma) as an HCV core binding protein by using yeast two-hybrid system. This interaction was demonstrated not only in cell culture but also in the livers of HCV core transgenic mice. These findings are extended to human HCV infection by the observation of this interaction in liver specimens from a patient with chronic HCV infection. Neither the interaction of HCV core protein with other PA28 subtypes nor that of PA28gamma with other Flavivirus core proteins was detected. Deletion of the PA28gamma-binding region from the HCV core protein or knockout of the PA28gamma gene led to the export of the HCV core protein from the nucleus to the cytoplasm. Overexpression of PA28gamma enhanced the proteolysis of the HCV core protein. Thus, the nuclear retention and stability of the HCV core protein is regulated via a PA28gamma-dependent pathway through which HCV pathogenesis may be exerted.