ATPase activity of p97/valosin-containing protein is regulated by oxidative modification of the evolutionally conserved cysteine 522 residue in Walker A motif

Valosin-containing protein (p97/VCP) has been proposed as playing crucial roles in a variety of physiological and pathological processes such as cancer and neurodegeneration. We previously showed that VCP(K524A), an ATPase activity-negative VCP mutant, induced vacuolization, accumulation of ubiquitinated proteins, and cell death, phenotypes commonly observed in neurodegenerative disorders. However, any regulatory mechanism of its ATPase activity has not yet been clarified. Here, we show that oxidative stress readily inactivates VCP ATPase activity. With liquid chromatography/tandem mass spectrometry, we found that at least three cysteine residues were modified by oxidative stress. Of them, the 522nd cysteine (Cys-522) was identified as the site responsible for the oxidative inactivation of VCP. VCP(C522T), a single-amino acid substitution mutant from cysteine to threonine, conferred almost complete resistance to the oxidative inactivation. In response to oxidative stress, VCP strengthened the interaction with Npl4 and Ufd1, both of which are essential in endoplasmic reticulum-associated protein degradation. Cys-522 is located in the second ATP binding motif and is highly conserved in multicellular but not unicellular organisms. Cdc48p (yeast VCP) has threonine in the corresponding amino acid, and it showed resistance to the oxidative inactivation in vitro. Furthermore, a yeast mutant (delta cdc48 + cdc48[T532C]) was shown to be susceptible to oxidants-induced growth inhibition and cell death. These results clearly demonstrate that VCP ATPase activity is regulated by the oxidative modification of the Cys-522 residue. This regulatory mechanism may play a key role in the conversion of oxidative stress to endoplasmic reticulum stress response in multicellular organisms and also in the pathological process of various neurodegenerative disorders.     

Time-related mapping of quantitative trait loci controlling grain-filling in rice (Oryza sativa L.)

Grain-filling is a crucial process that determines final grain yield in rice (Oryza sativa L.). To understand the genetic basis of dynamics of grain-filling, quantitative trait locus (QTL) analysis was conducted using time-related phenotypic data on grain-filling collected from a population of 155 recombinant inbred lines (F12), derived from a cross between Milyang 23 and Akihikari. Two QTLs detected on chromosomes 8 and 12 were strongly associated with increased filling percentage per panicle. These QTLs were not linked with those controlling spikelet numbers per panicle. This result confers the possibility of improving grain-filling together with an enlargement of sink size. The QTL for filling percentage per panicle on chromosome 8 exactly overlapped that for non-structural carbohydrate (NSC) content in the culm and leaf sheaths during grain-filling, and the Milyang 23 allele associated with increased grain-filling percentage per panicle was associated with decreased NSC content. Therefore, this QTL may be directly involved in NSC translocation from the culm and leaf sheaths to panicle. In addition, the Milyang 23 alleles of QTLs associated with greater spikelet number per panicle on chromosomes 1 and 6 were also related with a reduction in NSC content in the culm and leaf sheaths during grain-filling. These results indicate that NSC dynamics during grain-filling is partly dependent on sink size. NSC accumulation in the culm and leaf sheaths at the heading stage was mainly controlled by different genetic regulations from NSC dynamics during grain-filling. Nitrogen dynamics during grain-filling may also be involved in carbohydrate dynamics.     

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.     

Caissarolysin I (Bcs I), a new hemolytic toxin from the Brazilian sea anemone Bunodosoma caissarum: Purification and biological characterization

Two cationic proteins, C1 and C3, were purified to homogeneity from the hemolytic fraction of the venom of Bunodosoma caissarum sea anemone. The purification processes employed gel filtration followed by ion exchange chromatography, being the purity and molecular mass confirmed by SDS-PAGE and mass spectrometry. Protein C1 represented the second major peak of the hemolytic fraction and was previously believed to be a cytolysin belonging to a new class of hemolysins. The C1 protein has a molecular mass of 15495 Da and was assayed for hemolysis, PLA₂ activity and acute toxicity in crabs and mice, showing no activity in these assays. It has an amino terminal with no similarity to all known hemolysins and, therefore, should not be considered a toxin, being its function completely unknown. The protein C3 (19757 Da), that also lacks PLA₂ activity, was recognized by antiserum against Eqt II and presented high hemolytic activity to human erythrocytes (ED₅₀ of 0.270 μg/ml), being named Caissarolysin I (Bcs I). Its activity was inhibited by pre-incubation with sphingomyelin (SM) and also when in presence of erythrocytes pre-treated with the SMase P2, a phospholipase D from the brown spider Loxosceles intermedia, indicating that SM is the main target of Bcs I. Caissarolysin I is the first hemolysin purified from a sea anemone belonging to the genus Bunodosoma and belongs to the Actinoporin family of sea anemone hemolysins.     

Stability of protease in organic solvent: structural identification by solid-state NMR of lyophilized papain before and after 1-propanol treatment and the corresponding enzymatic activities

Lyophilized enzyme powder is often used in organic solvents. However, the enzymatic activity decreases during the reaction process. In the present study, the relation between structural stability and enzymatic activity in an organic solvent was investigated. 13C cross-polarization magic angle spinning NMR spectroscopy was used to determine the secondary structure of lyophilized papain in the solid-state. Deconvolution of the peaks of the backbone carbonyl carbons suggested that the proportion of beta-sheet conformation increased after lyophilization from a phosphate buffer solution. The esterification of N-benzyloxycarbonyl phenylalanylalanine amide was attempted using the lyophilized papain as a catalyst in anhydrous 1-propanol. The yield of ester was 46.1% after 48 h at 50 degrees C, but this reaction slowed remarkably after 48 h. When the lyophilized papain was suspended in anhydrous 1-propanol for 7 days without the substrate, the proportion of beta-sheet conformation was further increased and the suspended papain had no activity. These results suggest that the increase in beta-sheet conformation caused inactivation of papain. The increase in beta-sheet conformation caused by both lyophilization and suspension in propanol was found, which was related to a decrease in enzymatic activity.     

Zaprinast, a well-known cyclic guanosine monophosphate-specific phosphodiesterase inhibitor, is an agonist for GPR35

We found that zaprinast, a well-known cyclic guanosine monophosphate-specific phosphodiesterase inhibitor, acted as an agonist for a G protein-coupled receptor, GPR35. In our intracellular calcium mobilization assay, zaprinast activated rat GPR35 strongly (geometric mean EC(50) value of 16nM), whereas it activated human GPR35 moderately (geometric mean EC(50) value of 840nM). We also demonstrated that GPR35 acted as a Galpha(i/o)- and Galpha(16)-coupled receptor for zaprinast when heterologously expressed in human embryonic kidney 293 (HEK 293) cells. These findings will facilitate the research on GPR35 and the drug discovery of the GPR35 modulators.     

Role of numb in dendritic spine development with a Cdc42 GEF intersectin and EphB2

Numb has been implicated in cortical neurogenesis during nervous system development, as a result of its asymmetric partitioning and antagonizing Notch signaling. Recent studies have revealed that Numb functions in clathrin-dependent endocytosis by binding to the AP-2 complex. Numb is also expressed in postmitotic neurons and plays a role in axonal growth. However, the functions of Numb in later stages of neuronal development remain unknown. Here, we report that Numb specifically localizes to dendritic spines in cultured hippocampal neurons and is implicated in dendritic spine morphogenesis, partially through the direct interaction with intersectin, a Cdc42 guanine nucleotide exchange factor (GEF). Intersectin functions as a multidomain adaptor for proteins involved in endocytosis and cytoskeletal regulation. Numb enhanced the GEF activity of intersectin toward Cdc42 in vivo. Expression of Numb or intersectin caused the elongation of spine neck, whereas knockdown of Numb and Numb-like decreased the protrusion density and its length. Furthermore, Numb formed a complex with EphB2 receptor-type tyrosine kinase and NMDA-type glutamate receptors. Knockdown of Numb suppressed the ephrin-B1-induced spine development and maturation. These results highlight a role of Numb for dendritic spine development and synaptic functions with intersectin and EphB2.     

Construction of engineered fructosyl peptidyl oxidase for enzyme sensor applications under normal atmospheric conditions

Current enzymatic methods for the analysis of glycated proteins use flavoenzymes that catalyze the oxidative deglycation of fructosyl peptides, designated as fructosyl peptidyl oxidases (FPOXs). However, as FPOXs are oxidases, the signals derived from electron mediator-type electrochemical monitoring based on them are affected by dissolved O₂. Improvement of dye-mediated dehydrogenase activity of FPOXs and its application to enzyme electrode construction were therefore undertaken. Saturation mutagenesis study on Asn56 of FPOX from Phaeosphaeria nodorum, produced mutants with marked decreases in the catalytic ability to employ O₂ as the electron acceptor, while showing higher dye-mediated dehydrogenase activity employing artificial electron acceptors than the parental enzyme. Thus constructed virtually fructosyl peptide dehydrogenase, Asn56Ala, was then applied to produce an enzyme electrode for the measurement of fructosyl-^α N-valyl-histidine (f-^αVal-His), the protease-digested product of HbA1c. The enzyme electrode could measure f-^αVal-His in the physiological target range in air.