Characterization of the denatured structure of pyrrolidone carboxyl peptidase from a hyperthermophile under nondenaturing conditions: role of the C-terminal alpha-helix of the protein in folding and stability

The cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from a hyperthermophile, Pyrococcus furiosus, can be trapped in the denatured state under nondenaturing conditions, corresponding to the denatured structure that exists in equilibrium with the native state under physiological conditions. The denatured state is the initial state (D1 state) in the refolding process but differs from the completely denatured state (D2 state) in the concentrated denaturant. Also, it has been found that the D1 state corresponds to the heat-denatured state. To elucidate the structural basis of the D1 state, H/D exchange experiments with PCP-0SH were performed at pD 3.4 and 4 degrees C. The results indicated that amide protons in the C-terminal alpha6-helix region hardly exchanged in the D1 state with deuterium even after 7 days, suggesting that the alpha6-helix (from Ser188 to Glu205) of PCP-0SH was stably formed in the D1 state. In order to examine the role of the alpha6-helix in folding and stability, H/D exchange experiments with a mutant, A199P, at position 199 in the alpha6-helix region were performed. The alpha6-helix region of A199P in the D1 state was partially unprotected, while some hydrophobic residues were protected against the H/D exchange, although these hydrophobic residues were unprotected in the wild-type protein. These results suggest that the structure of A199P in the D1 state formed a temporary stable denatured structure with a non-native hydrophobic cluster and the unstructured alpha6-helix. Both the stability and the refolding rate decreased by the substitution of Pro for Ala199. We can conclude that the native-like helix (alpha6-helix) of PCP-0SH is already constructed in the D1 state and is necessary for efficient refolding into the native structure and stabilization of PCP-0SH.     

Genomic characterization of the filamentous integrative bacteriophages {phi}RSS1 and {phi}RSM1, which infect Ralstonia solanacearum

The genomic DNA sequences were determined for two filamentous integrative bacteriophages, phiRSS1 and phiRSM1, of the phytopathogen Ralstonia solanacearum. The 6,662-base sequence of phiRSS1 contained 11 open reading frames (ORFs). In the databases, this sequence showed high homology (95% identity) to the circular double-stranded DNA plasmid pJTPS1 (6,633 bp) isolated from a spontaneously occurring avirulent mutant of R. solanacearum. Two major differences between the two sequences were observed within phiRSS1 ORF7, corresponding to pIII, a minor coat protein required for host adsorption, and at the phiRSS1 intergenic (IG) region. The 9,004-base sequence of phiRSM1 showed 12 ORFs located on the same strand (plus strand) and 2 ORFs on the opposite strand. Compared with Ff-type phages, two insertions are obvious in the phiRSM1 replication module. Genomic DNA fragments containing the phiRSM integration junctions were cloned and sequenced from phiRSM lysogenic strain R. solanacearum MAFF211270. The att core sequence was identified as 5'-TGGCGGAGAGGGT-3', corresponding to the 3' end of the serine tRNA (UCG) gene. Interestingly, ORF14, located next to the attP site on the phiRSM1 genome, showed high amino acid sequence homology with bacterial DNA recombinases and resolvases, different from XerCD recombinases. attP of phiRSS1 is within a sequence element of the IG region.     

Involvement of sphingosine 1-phosphate, a platelet-derived bioactive lipid, in contraction of mesangium cells

Platelet-derived mediators may play an important role in the development of renal diseases through interaction with glomerular mesangial cells (MCs), and we, in this study, examined the effect of sphingosine 1-phosphate (Sph-1-P), a bioactive lipid released from activated platelets, on the contraction of MCs. Sph-1-P was found to induce MC contraction through mediation of Rho kinase both in cell shape change and collagen gel contraction assays. The specific antagonist of the Sph-1-P receptor S1P(2) inhibited the response. Similar results were obtained when the supernatant from activated platelet suspensions were used instead of Sph-1-P. Our findings suggest that platelet-derived Sph-1-P may be involved in MC contraction via S1P(2) and that regulation of this receptor might be useful therapeutically.     

A recurrent mutation in type II collagen gene causes Legg-Calvé-Perthes disease in a Japanese family

Legg-Calvé-Perthes disease (LCPD) is a common childhood hip disorder characterized by sequential stages of involvement of the capital femoral epiphyses, including subchondral fracture, fragmentation, re-ossification and healing with residual deformity. Most cases are sporadic, but familial cases have been described, with some families having multiple affected members. Genetic factors have been implicated in the etiology of LCPD, but the causal gene has not been identified. We have located a missense mutation (p.G1170S) in the type II collagen gene (COL2A1) in a Japanese family with an autosomal dominant hip disorder manifesting as LCPD and showing considerable intra-familial phenotypic variation. This is the first report of a mutation in hereditary LCPD. COL2A1 mutations may be more common in LCPD patients than currently thought, particularly in familial and/or bilateral cases.     

Mycophenolic acid as a latent agonist of PPARgamma

Mycophenolic acid (MPA), known as an inhibitor of inosine monophosphate dehydrogenase (IMPDH), was found to inhibit the differentiation of 3T3-L1 pre-adipocytes into mature adipocytes. Although the effect of MPA was attributed to inhibition of IMPDH, we uncovered a hidden biological property of MPA as an agonist of peroxisome proliferator activated receptor gamma (PPARgamma).     

Discovery of (-)-6-[2-[4-(3-fluorophenyl)-4-hydroxy-1-piperidinyl]-1-hydroxyethyl]-3,4-dihydro-2(1H)-quinolinone--a potent NR2B-selective N-methyl D-aspartate (NMDA) antagonist for the treatment of pain

(-)-6-[2-[4-(3-Fluorophenyl)-4-hydroxy-1-piperidinyl]-1-hydroxyethyl]-3,4-dihydro-2(1H)-quinolinone was identified as an orally active NR2B-subunit selective N-methyl-d-aspartate (NMDA) receptor antagonist. It has very high selectivity for NR2B subunits containing NMDA receptors versus the HERG-channel inhibition (therapeutic index=4200 vs NR2B binding IC(50)). This compound has improved pharmacokinetic properties compared to the prototype CP-101,606.     

Role of Septin cytoskeleton in spine morphogenesis and dendrite development in neurons

Septins are GTP-binding proteins that polymerize into heteromeric filaments and form microscopic bundles or ring structures in vitro and in vivo. Because of these properties and their ability to associate with membrane, F-actin, and microtubules, septins have been generally regarded as cytoskeletal components [1, 2]. Septins are known to play roles in cytokinesis, in membrane trafficking, and as structural scaffolds; however, their function in neurons is poorly understood. Many members of the septin family, including Septin 7 (Sept7), were found by mass-spectrometry analysis of postsynaptic density (PSD) fractions of the brain [3, 4], suggesting a possible postsynaptic function of septins in neurons. We report that Sept7 is localized at the base of dendritic protrusions and at dendritic branch points in cultured hippocampal neurons--a distribution reminiscent of septin localization in the bud neck of budding yeast. Overexpression of Sept7 increased dendrite branching and the density of dendritic protrusions, whereas RNA interference (RNAi)-mediated knockdown of Sept7 led to reduced dendrite arborization and a greater proportion of immature protrusions. These data suggest that Sept7 is critical for spine morphogenesis and dendrite development during neuronal maturation.