Dual subcellular localization in the endoplasmic reticulum and peroxisomes and a vital role in protecting against oxidative stress of fatty aldehyde dehydrogenase are achieved by alternative splicing

Fatty aldehyde dehydrogenase (FALDH, ALDH3A2) is thought to be involved in the degradation of phytanic acid, a saturated branched chain fatty acid derived from chlorophyll. However, the identity, subcellular distribution, and physiological roles of FALDH are unclear because several variants produced by alternative splicing are present in varying amounts at different subcellular locations. Subcellular fractionation experiments do not provide a clear-cut conclusion because of the incomplete separation of organelles. We established human cell lines heterologously expressing mouse FALDH from each cDNA without tagging under the control of an inducible promoter and detected the variant FALDH proteins using a mouse FALDH-specific antibody. One variant, FALDH-V, was exclusively detected in peroxisomal membranes. Human FALDH-V with an amino-terminal Myc sequence also localized to peroxisomes. The most dominant form, FALDH-N, and other variants examined, however, were distributed in the endoplasmic reticulum. A gas chromatography-mass spectrometry-based analysis of metabolites in FALDH-expressing cells incubated with phytol or phytanic acid showed that FALDH-V, not FALDH-N, is the key aldehyde dehydrogenase in the degradation pathway and that it protects peroxisomes from oxidative stress. In contrast, both FALDHs had a protective effect against oxidative stress induced by a model aldehyde for lipid peroxidation, dodecanal. These results suggest that FALDH variants are produced by alternative splicing and share an important role in protecting against oxidative stress in an organelle-specific manner.     

Doublecortin microtubule affinity is regulated by a balance of kinase and phosphatase activity at the leading edge of migrating neurons

Doublecortin (Dcx) is a microtubule-associated protein that is mutated in X-linked lissencephaly (X-LIS), a neuronal migration disorder associated with epilepsy and mental retardation. Although Dcx can bind ubiquitously to microtubules in nonneuronal cells, Dcx is highly enriched in the leading processes of migrating neurons and the growth cone region of differentiating neurons. We present evidence that Dcx/microtubule interactions are negatively controlled by Protein Kinase A (PKA) and the MARK/PAR-1 family of protein kinases. In addition to a consensus MARK site, we identified a serine within a novel sequence that is crucial for the PKA- and MARK-dependent regulation of Dcx's microtubule binding activity in vitro. This serine is mutated in two families affected by X-LIS. Immunostaining neurons with an antibody that recognizes phosphorylated substrates of MARK supports the conclusion that Dcx localization and function are regulated at the leading edge of migrating cells by a balance of kinase and phosphatase activity.     

Long-lasting smooth-muscle relaxation by a novel PACAP analogue in human bronchi

We compared the relaxant effect of original pituitary adenylate cyclase-activating peptide (PACAP)1-27 with that of a newly developed, synthetic PACAP1-27 analogue, [Arg15,20,21 Leu17]-PACAP-Gly-Lys-Arg-NH2, in human bronchi in vitro (n=4-5 in each group). Using precontraction by carbachol (0.1 microM), cumulative administration of PACAP1-27 and salbutamol caused concentration-dependent smooth muscle relaxation with similar potencies and maximum relaxant effects. Non-cumulative administration of the PACAP1-27 analogue and the original PACAP1-27 caused concentration-dependent relaxation with a similar maximum relaxant effect and potency as well. However, the onset and offset of action was markedly slower for the PACAP1-27 analogue than for the original PACAP1-27 (>90% versus <10% of peak relaxation remaining 5 h after administration). Peptidase inhibition by captopril (10 microM) and phosphoramidon (1 microM) significantly increased the maximum relaxant effect and duration of action of PACAP1-27 but not of the PACAP1-27 analogue, during the 3 h of observation in the human bronchi. We conclude that [Arg15,20,21 Leu17]-PACAP-Gly-Lys-Arg-NH2 produces significant concentration-dependent and sustained bronchial smooth muscle relaxation in vitro. The sustained relaxant effect is due, at least in part, to the synthetic PACAP1-27 analogue being less susceptible to cleavage by peptidases than the original peptide PACAP1-27.     

Generation of reactive oxygen species undergoing redox cycle of nostocine A: a cytotoxic violet pigment produced by freshwater cyanobacterium Nostoc spongiaeforme

Nostocine A (1) is an extracellular cytotoxic violet pigment produced by the freshwater cyanobacterium, Nostoc spongiaeforme TISTR 8169. Treatment with 1 was found to accelerate the generation of reactive oxygen species (ROS) in the green alga, Chlamydomonas reinhardtii, in the light. In vitro analysis revealed that 1 specifically eliminated superoxide radical anion (O(2)(-)) among several ROS tested. During the course of the reaction, oxygen (O(2)) was simultaneously synthesized and the O(2) synthesizing rate increased with the amount of 1 added. In contrast, O(2)(-) generation occurred when NADPH or NADH was added to a solution of 1 under aerobic condition. The reduction potential of 1 is very similar to that of O(2) indicating that 1 and O(2) can easily exchange electrons depending on the mass balance between their oxidized and reduced forms. Based on these results, the following hypothesis is formulated for the mechanism of intracellular ROS generation by treatment with 1: 1 taken into the target cells is reduced specifically by intracellular reductants such as NAD(P)H. When the O(2) level is sufficiently higher than that of 1, the reduced product of 1 is immediately oxidized by O(2). This is accompanied by the synthesis of O(2)(-) from O(2). The generation of O(2)(-) successively occurs, undergoing repeated redox cycles of 1, when the levels of the reductant and O(2) are still dominant to promote these reactions. This similar intracellular ROS generation mechanism to that of paraquat may cause the cytotoxicity.     

BIG2, a guanine nucleotide exchange factor for ADP-ribosylation factors: its localization to recycling endosomes and implication in the endosome integrity

Small GTPases of the ADP-ribosylation factor (ARF) family play a key role in membrane trafficking by regulating coated vesicle formation, and guanine nucleotide exchange is essential for the ARF function. Brefeldin A blocks the ARF-triggered coat assembly by inhibiting the guanine nucleotide exchange on ARFs and causes disintegration of the Golgi complex and tubulation of endosomal membranes. BIG2 is one of brefeldin A-inhibited guanine nucleotide exchange factors for the ARF GTPases and is associated mainly with the trans-Golgi network. In the present study, we have revealed that another population of BIG2 is associated with the recycling endosome and found that expression of a catalytically inactive BIG2 mutant, E738K, selectively induces membrane tubules from this compartment. We also have shown that BIG2 has an exchange activity toward class I ARFs (ARF1 and ARF3) in vivo and inactivation of either ARF exaggerates the BIG2(E738K)-induced tubulation of endosomal membranes. These observations together indicate that BIG2 is implicated in the structural integrity of the recycling endosome through activating class I ARFs.     

Recombinant alkaline serine protease II degrades scrapie isoform of prion protein

Summary An efficient Escherichia coli expression system for the production of mature-type alkaline serine protease II (mASP II) has been constructed. Complementary deoxyribonucleic acid-encoding mASP II was inserted into the inducible bacterial expression vector pGE-30. After introduction into E, coli, the plasmid was expressed by isopropyl-1-thio-β-d-galactopyranoside, and the recombinant product was purified using a Ni-nitrilotriacetic acid column The purified product had the expected NH₂-terminal sequence and showed a scrapie isoform of prion protein-degrading activity using hamster scrapie 263K prions as a substrate.     

Sphingosine-dependent protein kinase-1, directed to 14-3-3, is identified as the kinase domain of protein kinase C delta

Some protein kinases are known to be activated by d-erythro-sphingosine (Sph) or N,N-dimethyl-d-erythro-sphingosine (DMS), but not by ceramide, Sph-1-P, other sphingolipids, or phospholipids. Among these, a specific protein kinase that phosphorylates Ser60, Ser59, or Ser58 of 14-3-3beta, 14-3-3eta, or 14-3-3zeta, respectively, was termed "sphingosine-dependent protein kinase-1" (SDK1) (Megidish, T., Cooper, J., Zhang, L., Fu, H., and Hakomori, S. (1998) J. Biol. Chem. 273, 21834-21845). We have now identified SDK1 as a protein having the C-terminal half kinase domain of protein kinase Cdelta (PKCdelta) based on the following observations. (i). Large-scale preparation and purification of proteins showing SDK1 activity from rat liver (by six steps of chromatography) gave a final fraction with an enhanced level of an approximately 40-kDa protein band. This fraction had SDK1 activity approximately 50000-fold higher than that in the initial extract. (ii). This protein had approximately 53% sequence identity to the Ser/Thr kinase domain of PKCdelta based on peptide mapping using liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry data. (iii). A search for amino acid homology based on the BLAST algorithm indicated that the only protein with high homology to the approximately 40-kDa band is the kinase domain of PKCdelta. The kinase activity of PKCdelta did not depend on Sph or DMS; rather, it was inhibited by these sphingoid bases, i.e. PKCdelta did not display any SDK1 activity. However, strong SDK1 activity became detectable when PKCdelta was incubated with caspase-3, which releases the approximately 40-kDa kinase domain. PKCdelta and SDK1 showed different lipid requirements and substrate specificity, although both kinase activities were inhibited by common PKC inhibitors. The high susceptibility of SDK1 to Sph and DMS accounts for their important modulatory role in signal transduction.     

A sphingosine-dependent protein kinase that specifically phosphorylates 14-3-3 (SDK1) is identified as the kinase domain of PKCdelta: a preliminary note

A specific protein kinase that phosphorylates Ser60, Ser59, or Ser58 of 14-3-3beta, eta, or zeta, respectively, only in the presence of sphingosine (Sph) or N,N-dimethyl-Sph (DMS), was termed "sphingosine-dependent protein kinase-1" (SDK1) [J. Biol. Chem. 273(34) (1998) 21834]. We have now identified SDK1 as a protein having the same amino acid sequence as in the C-terminal-half kinase domain of PKCdelta, with approximately 40 kDa molecular mass, based on large-scale purification of a protein from rat liver, and partial sequence using three different combinations of LC-MS or LC-MS/MS with respective search engine. PKCdelta did not display any SDK1 activity and PKCdelta activity was inhibited by Sph and DMS. However, strong SDK1 activity, only in the presence of Sph or DMS, became detectable when PKCdelta was incubated with caspase-3, which releases the approximately 40 kDa kinase domain.