The Role of Individual Domains and the Significance of Shedding of ATP6AP2/(pro)renin Receptor in Vacuolar H+-ATPase Biogenesis

The ATPase 6 accessory protein 2 (ATP6AP2)/(pro)renin receptor (PRR) is essential for the biogenesis of active vacuolar H⁺-ATPase (V-ATPase). Genetic deletion of ATP6AP2/PRR causes V-ATPase dysfunction and compromises vesicular acidification. Here, we characterized the domains of ATP6AP2/PRR involved in active V-ATPase biogenesis. Three forms of ATP6AP2/PRR were found intracellularly: full-length protein and the N- and C-terminal fragments of furin cleavage products, with the N-terminal fragment secreted extracellularly. Genetic deletion of ATP6AP2/PRR did not affect the protein stability of V-ATPase subunits. The extracellular domain (ECD) and transmembrane domain (TM) of ATP6AP2/PRR were indispensable for the biogenesis of active V-ATPase. A deletion mutant of ATP6AP2/PRR, which lacks exon 4-encoded amino acids inside the ECD (Δ4M) and causes X-linked mental retardation Hedera type (MRXSH) and X-linked parkinsonism with spasticity (XPDS) in humans, was defective as a V-ATPase-associated protein. Prorenin had no effect on the biogenesis of active V-ATPase. The cleavage of ATP6AP2/PRR by furin seemed also dispensable for the biogenesis of active V-ATPase. We conclude that the N-terminal ECD of ATP6AP2/PRR, which is also involved in binding to prorenin or renin, is required for the biogenesis of active V-ATPase. The V-ATPase assembly occurs prior to its delivery to the trans-Golgi network and hence shedding of ATP6AP2/PRR would not affect the biogenesis of active V-ATPase.     

Stimulation of Bone Formation in Cortical Bone of Mice Treated with a Receptor Activator of Nuclear Factor-κB Ligand (RANKL)-binding Peptide That Possesses Osteoclastogenesis Inhibitory Activity

To date, parathyroid hormone is the only clinically available bone anabolic drug. The major difficulty in the development of such drugs is the lack of clarification of the mechanisms regulating osteoblast differentiation and bone formation. Here, we report a peptide (W9) known to abrogate osteoclast differentiation in vivo via blocking receptor activator of nuclear factor-κB ligand (RANKL)-RANK signaling that we surprisingly found exhibits a bone anabolic effect in vivo. Subcutaneous administration of W9 three times/day for 5 days significantly augmented bone mineral density in mouse cortical bone. Histomorphometric analysis showed a decrease in osteoclastogenesis in the distal femoral metaphysis and a significant increase in bone formation in the femoral diaphysis. Our findings suggest that W9 exerts bone anabolic activity. To clarify the mechanisms involved in this activity, we investigated the effects of W9 on osteoblast differentiation/mineralization in MC3T3-E1 (E1) cells. W9 markedly increased alkaline phosphatase (a marker enzyme of osteoblasts) activity and mineralization as shown by alizarin red staining. Gene expression of several osteogenesis-related factors was increased in W9-treated E1 cells. Addition of W9 activated p38 MAPK and Smad1/5/8 in E1 cells, and W9 showed osteogenesis stimulatory activity synergistically with BMP-2 in vitro and ectopic bone formation. Knockdown of RANKL expression in E1 cells reduced the effect of W9. Furthermore, W9 showed a weak effect on RANKL-deficient osteoblasts in alkaline phosphatase assay. Taken together, our findings suggest that this peptide may be useful for the treatment of bone diseases, and W9 achieves its bone anabolic activity through RANKL on osteoblasts accompanied by production of several autocrine factors.     

Reconstitution of Homomeric GluA2flop Receptors in Supported Lipid Membranes: FUNCTIONAL AND STRUCTURAL PROPERTIES*

AMPA receptors (AMPARs) are glutamate-gated ion channels ubiquitous in the vertebrate central nervous system, where they mediate fast excitatory neurotransmission and act as molecular determinants of memory formation and learning. Together with detailed analyses of individual AMPAR domains, structural studies of full-length AMPARs by electron microscopy and x-ray crystallography have provided important insights into channel assembly and function. However, the correlation between the structure and functional states of the channel remains ambiguous particularly because these functional states can be assessed only with the receptor bound within an intact lipid bilayer. To provide a basis for investigating AMPAR structure in a membrane environment, we developed an optimized reconstitution protocol using a receptor whose structure has previously been characterized by electron microscopy. Single-channel recordings of reconstituted homomeric GluA2_flop receptors recapitulate key electrophysiological parameters of the channels expressed in native cellular membranes. Atomic force microscopy studies of the reconstituted samples provide high-resolution images of membrane-embedded full-length AMPARs at densities comparable to those in postsynaptic membranes. The data demonstrate the effect of protein density on conformational flexibility and dimensions of the receptors and provide the first structural characterization of functional membrane-embedded AMPARs, thus laying the foundation for correlated structure-function analyses of the predominant mediators of excitatory synaptic signals in the brain.     

Cysteine 295 indirectly affects Ni coordination of carbon monoxide dehydrogenase-II C-cluster

A unique [Ni–Fe–S] cluster (C-cluster) constitutes the active center of Ni-containing carbon monoxide dehydrogenases (CODHs). His²⁶¹, which coordinates one of the Fe atoms with Cys²⁹⁵, is suggested to be the only residue required for Ni coordination in the C-cluster. To evaluate the role of Cys²⁹⁵, we constructed CODH-II variants. Ala substitution for the Cys²⁹⁵ substitution resulted in the decrease of Ni content and didn’t result in major change of Fe content. In addition, the substitution had no effect on the ability to assemble a full complement of [Fe–S] clusters. This strongly suggests Cys²⁹⁵ indirectly and His²⁶¹ together affect Ni-coordination in the C-cluster.     

The binding of okadaic acid analogs to recombinant OABP2.1 originally isolated from the marine sponge Halichondria okadai

The binding between [24-³H]okadaic acid (OA) and a recombinant OA binding protein OABP2.1 was examined using various OA analog, including methyl okadaate, norokadanone, 7-deoxy OA, and 14,15-dihydro OA, 7-O-palmitoyl DTX1, to investigate the structure activity relationship. Among them, 7-O-palmitoyl DTX1, which is one of the diarrhetic shellfish poisoning (DSP) toxins identified in shellfish, displayed an IC₅₀ for [24-³H]OA binding at 51 ± 6.3 nM (Mean ± SD). In addition, a synthetic compound, N-pyrenylmethyl okadamide, exhibited its IC₅₀ at 10 ± 2.9 nM (Mean ± SD). These results suggested that the recombinant OABP2.1 and the N-pyrenylmethyl okadamide might be core substances in a novel assay for the DSP toxins.     

Interactions of bacterial flagellar chaperone–substrate complexes with FlhA contribute to co‐ordinating assembly of the flagellar filament

Assembly of the bacterial flagellar filament is strictly sequential; the junction proteins, FlgK and FlgL, are assembled at the distal end of the hook prior to the FliD cap, which supports assembly of as many as 30 000 FliC molecules into the filament. Export of these proteins requires assistance of flagellar chaperones: FlgN for FlgK and FlgL, FliT for FliD and FliS for FliC. The C‐terminal cytoplasmic domain of FlhA (FlhA_C), a membrane component of the export apparatus, provides a binding‐site for these chaperone–substrate complexes but it remains unknown how it co‐ordinates flagellar protein export. Here, we report that the highly conserved hydrophobic dimple of FlhA_C is involved in the export of FlgK, FlgL, FliD and FliC but not in proteins responsible for the structure and assembly of the hook, and that the binding affinity of FlhA_C for the FlgN/FlgK complex is slightly higher than that for the FliT/FliD complex and about 14‐fold higher than that for the FliS/FliC complex, leading to the proposal that the different binding affinities of FlhA_C for these chaperone/substrate complexes may confer an advantage for the efficient formation of the junction and cap structures at the tip of the hook prior to filament formation.     

Phytotoxins Produced by Onion Pink Root Fungus,Pyrenochaeta terrestris

Dextranase (EC 3.2.1.11) produced by Chaetomium gracile was purified by sequential chromatographies on CM- and DEAE-cellulose columns, and two active fractions, CD-I and CD-II, were isolated in electrophoretically pure states. The former fraction was obtained in a crystalline state. The estimated molecular weights were 77,000 for CD-I and 71,000 for CD-II, and their isoelectric points were 6.2 and 5.7, respectively. Both active fractions contained a sugar moiety (4.5%). Their amino acid compositions were determined. They were very similar to each other in enzymatic properties: The optimum pH was at around 5.5, and they were stable between pH 5.5 and 11.0, and at temperatures lower than 55°C. They were typical endodextranases, but their maximal degrees of dextran hydrolysis reached 55% as glucose.     

Studies on the Metabolic Products of Oospora sp.

A microorganism was isolated from the air of a patient-room and classified in the genus Oospora. This microorganism was cultured on a malt extract medium, and the mycellium was separated from the culture filtrate. A new compound (O-1), m.p. 129°C, C₁₁H₁₀O₃, and eburicoic acid, m.p. 290°C, C₃₁H₅₀O₃ were obtained from the dried mycellium. Another new compound (O-2), m.p. 176°C, C₁₁H₈O₅ was obtained from the culture filtrate.     

X-Ray Crystal Structure of Pyrenocine A,a Phytotoxin from Pyrenochaeta terrestris

The milk fat globule membrane (MFGM) enclosing fat droplets in bovine milk was isolated, and its effects on hydrolysis of milk fat by lipases were investigated by using a gum arabic-stabilized milk fat emulsion as substrate. The addition of isolated MFGM to the reaction mixture markedly inhibited hydrolysis by pancreatic and microbial (Rhizopus delemer) lipases. The inhibition was completely lost on tryptic digestion of MFGM, suggesting that the protein moiety of MFGM played a role in the inhibition. Soluble glycoprotein (SGP) which was isolated from delipidated MFGM produced marked inhibitory activity. The inhibition by SGP was dependent on substrate concentration, suggesting that the inhibition was at least partly due to coverage and blockage of the substrate surface by SGP.