Neurotrophic activity of neudesin, a novel extracellular heme-binding protein, is dependent on the binding of heme to its cytochrome b5-like heme/steroid-binding domain

Neudesin is a secreted protein with neurotrophic activity in neurons and undifferentiated neural cells. We report here that neudesin is an extracellular heme-binding protein and that its neurotrophic activity is dependent on the binding of heme to its cytochrome b(5)-like heme/steroid-binding domain. At first, we found that at least a portion of the purified recombinant neudesin appeared to bind hemin because the purified neudesin solution was tinged with green and had a sharp absorbance peak at 402 nm. The addition of exogenous hemin extensively increased the amount of hemin-bound neudesin. In contrast, neudesinDeltaHBD, a mutant lacking the heme-binding domain, could not bind hemin. The neurotrophic activity of the recombinant neudesin that bound exogenous hemin (neudesin-hemin) was significantly greater than that of the recombinant neudesin in either primary cultured neurons or Neuro2a cells, suggesting that the activity of neudesin depends on hemin. The neurotrophic activity of neudesin was enhanced by the binding of Fe(III)-protoporphyrin IX, but neither Fe(II)-protoporphyrin IX nor protoporphyrin IX alone. The inhibition of endogenous neudesin by RNA interference significantly decreased cell survival in Neuro2a cells. This indicates that endogenous neudesin possibly contains hemin. The experiment with anti-neudesin antibody suggested that the endogenous neudesin detected in the culture medium of Neuro2a cells was associated with hemin because it was not retained on a heme-affinity column at all. Neudesin is the first extracellular heme-binding protein that shows signal transducing activity by itself. The present findings may shed new light on the function of extracellular heme-binding proteins.     

Double deficiency of tetraspanins CD9 and CD81 alters cell motility and protease production of macrophages and causes chronic obstructive pulmonary disease-like phenotype in mice

CD9 and CD81 are closely related tetraspanins that regulate cell motility and signaling by facilitating the organization of multimolecular membrane complexes, including integrins. We show that CD9 and CD81 are down-regulated in smoking-related inflammatory response of a macrophage line, RAW264.7. When functions of CD9 and CD81 were ablated with monoclonal antibody treatment, small interfering RNA transfection, or gene knock-out, macrophages were less motile and produced larger amounts of matrix metalloproteinase (MMP)-2 and MMP-9 than control cells in vitro. In line with this, CD9/CD81 double-knock-out mice spontaneously developed pulmonary emphysema, a major pathological component of chronic obstructive pulmonary disease (COPD). The mutant lung contained an increased number of alveolar macrophages with elevated activities of MMP-2 and MMP-9 and progressively displayed enlarged airspace and disruption of elastic fibers in the alveoli. Secretory cell metaplasia, a finding similar to goblet cell metaplasia in cigarette smokers, was also observed in the epithelium of terminal bronchioles. With aging, the double-knockout mice showed extrapulmonary phenotypes, including weight loss, kyphosis, and osteopenia. These results suggest that the tetraspanins CD9 and CD81 regulate cell motility and protease production of macrophages and that their dysfunction may underlie the progression of COPD.     

Dissection of the essential steps for condensin accumulation at kinetochores and rDNAs during fission yeast mitosis

The condensin complex has a fundamental role in chromosome dynamics. In this study, we report that accumulation of Schizosaccharomyces pombe condensin at mitotic kinetochores and ribosomal DNAs (rDNAs) occurs in multiple steps and is necessary for normal segregation of the sister kinetochores and rDNAs. Nuclear entry of condensin at the onset of mitosis requires Cut15/importin alpha and Cdc2 phosphorylation. Ark1/aurora and Cut17/Bir1/survivin are needed to dock the condensin at both the kinetochores and rDNAs. Furthermore, proteins that are necessary to form the chromatin architecture of the kinetochores (Mis6, Cnp1, and Mis13) and rDNAs (Nuc1 and Acr1) are required for condensin to accumulate specifically at these sites. Acr1 (accumulation of condensin at rDNA 1) is an rDNA upstream sequence binding protein that physically interacts with Rrn5, Rrn11, Rrn7, and Spp27 and is required for the proper accumulation of Nuc1 at rDNAs. The mechanism of condensin accumulation at the kinetochores may be conserved, as human condensin II fails to accumulate at kinetochores in hMis6 RNA interference-treated cells.     

A betacellulin mutant promotes differentiation of pancreatic acinar AR42J cells into insulin-producing cells with low affinity of binding to ErbB1

Betacellulin (BTC) is one of the members of the epidermal growth factor (EGF) ligand family of ErbB receptor tyrosine kinases. It is a differentiation factor as well as a potent mitogen. BTC promotes the differentiation of pancreatic acinar-derived AR42J cells into insulin-producing cells. It independently and preferentially binds to two type I tyrosine kinase receptors, the EGF receptor (ErbB1) and ErbB4. However, the physiochemical characteristics of BTC that are responsible for its preferential binding to these two receptors have not been fully defined. In this study, to investigate the essential amino acid residues of BTC for binding to the two receptors, we introduced point mutations into the EGF domain of BTC employing error-prone PCR. The receptor binding abilities of 190 mutants expressed in Escherichia coli were assessed by enzyme immunoassay. Replacement of the glutamic acid residue at position 88 with a lysine residue in BTC was found to produce a significant loss of affinity for binding to ErbB1, while the affinity of binding to ErbB4 was unchanged. In addition, the mutant of BTC-E/88/K showed less growth-promoting activity on BALB/c 3T3 cells compared with that of the wild-type BTC protein. Interestingly, the BTC mutant protein promoted differentiation of pancreatic acinar AR42J cells at a high frequency into insulin-producing cells compared with AR42J cells that were treated with wild-type BTC protein. These results indicate the possibility of designing BTC mutants, which have an activity of inducing differentiation only, without facilitating growth promotion.     

Crystal structure of human ribosomal protein L10 core domain reveals eukaryote-specific motifs in addition to the conserved fold

A phylogenetically conserved ribosomal protein L16p/L10e organizes the architecture of the aminoacyl tRNA binding site on the large ribosomal subunit. Eukaryotic L10 also exhibits a variety of cellular activities, and, in particular, human L10 is known as a putative tumor suppressor, QM. We have determined the 2.5-Å crystal structure of the human L10 core domain that corresponds to residues 34-182 of the full-length 214 amino acids. Its two-layered α + β architecture is significantly similar to those of the archaeal and bacterial homologues, substantiating a high degree of structural conservation across the three phylogenetic domains. A cation-binding pocket formed between α2 and β6 is similar to that of the archaeal L10 protein but appears to be better ordered. Previously reported L10 mutations that cause defects in the yeast ribosome are clustered around this pocket, indicating that its integrity is crucial for its role in L10 function. Characteristic interactions among Arg90-Trp171-Arg139 guide the C-terminal part outside of the central fold, implying that the eukaryote-specific C-terminal extension localizes on the outer side of the ribosome.     

Imino proton NMR analysis of HDV ribozymes: nested double pseudoknot structure and Mg2+ ion-binding site close to the catalytic core in solution

Minimized trans-acting HDV ribozyme systems consisting of three (Rz-3) and two (Rz-2) RNA strands were prepared and their folding conformations were analyzed by NMR spectroscopy. The guanosine residues in one of the enzyme components of Rz-3 were labeled with ¹³C and ¹⁵N. Imino proton signals were assigned by analysis of NOESY and HSQC spectra. The results are consistent with the nested double pseudoknot model, which contains novel base pairs (P1.1), as observed in the crystal structure of a genomic HDV ribozyme. The NOE connectivities suggest an additional G:G pair at the bottom of P1.1 and at the top of P4. The effects of temperature and Mg²⁺ ions on base pairs for Rz-3 were examined. The temperature variation experiment on Rz-3 showed that P3 is the most stable and that P1.1 is as stable as P1 and P2. The imino proton signals of the G:U pair at the bottom of P1 and the top of P1.1, which are close to the cleavage site, showed the largest changes upon Mg²⁺ titration of Rz-3. The results suggest that the catalytic Mg²⁺ ion binds to the pocket formed by P1 and L3.     

Effects of ceramide,ceramidase inhibition and expression of ceramide kinase on cytosolic phospholipase A2α; additional role of ceramide-1-phosphate in phosphorylation and Ca2+ signaling

Ceramide and the metabolites including ceramide-1-phosphate (C1P) and sphingosine are reported to regulate the release of arachidonic acid (AA) and/or phospholipase A₂ (PLA₂) activity in many cell types including lymphocytes. Recent studies established that C1P, a product of ceramide kinase, interacts directly with Ca²⁺ binding regions in the C2 domain of α type cytosolic PLA₂ (cPLA₂α), leading to translocation of the enzyme from the cytosol to the perinuclear region in cells. However, a precise mechanism for C1P-induced activation of cPLA₂α has not been well elucidated; such as the phosphorylation signal caused by the extracellular signal-regulated kinases (ERK1/2) pathway, a downstream of the protein kinase C activation with 4β-phorbol myristate acetate (PMA), is required or not. In the present study, we showed that the increase in intracellular ceramide levels (exogenously added cell permeable ceramides and an inhibition of ceramidase by (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol and the increase in C1P formation by transfection with the vector for human ceramide kinase significantly enhanced the Ca²⁺ ionophore (A23187) -induced release of AA via cPLA₂α's activation in CHO cells. Ceramides did not show additional effects on the release from the cells treated with the inhibitor of ceramidase. Ceramides and C2-C1P neither had effect on the intracellular mobilization of Ca²⁺ nor the phosphorylation of cPLA₂α in cells. A23187/PMA-induced release of AA was enhanced by ceramides and C2-C1P and by expression of ceramide kinase. Our findings suggest that C1P is a stimulatory factor on cPLA₂α that is independent of the Ca²⁺ signal and the PKC-ERK-mediated phosphorylation signal.     

Alantrypinone and its derivatives: Synthesis and antagonist activity toward insect GABA receptors

The γ-aminobutyric acid (GABA) receptor bears important sites of action for insecticides. Alantrypinone is an insecticidal alkaloid that acts as a selective antagonist for housefly (vs rat) GABA receptors, and is considered to be a lead compound for the development of a safer insecticide. In an attempt to obtain compounds with greater activity, a series of racemic alantrypinone derivatives were systematically synthesized using hetero Diels–Alder reactions, and a total of 34 compounds were examined for their ability to inhibit the specific binding of [³H]4′-ethynyl-4-n-propylbicycloorthobenzoate, a high-affinity non-competitive antagonist, to housefly-head membranes. The assay results showed that (1) there is no significant difference between the potencies of natural (+)-alantrypinone and its synthetic racemate; (2) the amide NHs at the 2- and 18-positions are important for high activity; (3) there is a considerable drop in potency for compounds without an aromatic ring at the 16-position; and (4) a large substituent at the 3-position is detrimental to high activity.