Shoko Miyazawa

Amino-terminal presequence of the precursor of peroxisomal 3-ketoacyl-CoA thiolase is a cleavable signal peptide for peroxisomal targeting

To examine the function of the amino-terminal presequence of rat peroxisomal 3-ketoacyl-CoA thiolase precursor, fusion proteins of various amino-terminal regions of the precursor with non-peroxisomal enzymes were expressed in cultured mammalian cells. On immunofluorescence microscopy, all constructs carrying the presequence part exhibited punctate patterns of distribution, identical with that of catalase, a peroxisomal marker. Proteins lacking all or a part of the prepiece were found in the cytosol. These results indicate that the presequence of the thiolase has sufficient information for peroxisomal targeting.

Peroxisome targeting signal of rat liver acyl-coenzyme A oxidase resides at the carboxy terminus.

To identify the topogenic signal of peroxisomal acyl-coenzyme A oxidase (AOX) of rat liver, we carried out in vitro import experiments with mutant polypeptides of the enzyme. Full-length AOX and polypeptides that were truncated at the N-terminal region were efficiently imported into peroxisomes, as determined by resistance to externally added proteinase K. Polypeptides carrying internal deletions in the C-terminal region exhibited much lower import activities. Polypeptides that were truncated or mutated at the extreme C terminus were totally import negative. When the five amino acid residues at the extreme C terminus were attached to some of the import-negative polypeptides, the import activities were rescued. Moreover, the C-terminal 199 and 70 amino acid residues of AOX directed fusion proteins with two bacterial enzymes to peroxisomes. These results are interpreted to mean that the peroxisome targeting signal of AOX residues at the C terminus and the five or fewer residues at the extreme terminus have an obligatory function in targeting. The C-terminal internal region also has an important role for efficient import, possibly through a conformational effect.

d-3-Hydroxyacyl-CoA Dehydratase/d-3-Hydroxyacyl-CoA Dehydrogenase Bifunctional Protein Deficiency: A Newly Identified Peroxisomal Disorder

Peroxisomal beta-oxidation proceeds from enoyl-CoA through D-3-hydroxyacyl-CoA to 3-ketoacyl-CoA by the D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxy-acyl-CoA dehydrogenase bifunctional protein (d-bifunctional protein), and the oxidation of bile-acid precursors also has been suggested as being catalyzed by the d-bifunctional protein. Because of the important roles of this protein, we reinvestigated two Japanese patients previously diagnosed as having enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase bifunctional protein (L-bifunctional protein) deficiency, in complementation studies. We found that both the protein and the enzyme activity of the d-bifunctional protein were hardly detectable in these patients but that the active L-bifunctional protein was present. The mRNA level in patient 1 was very low, and, for patient 2, mRNA was of a smaller size. Sequencing analysis of the cDNA revealed a 52-bp deletion in patient 1 and a 237-bp deletion in patient 2. This seems to be the first report of D-bifunctional protein deficiency. Patients previously diagnosed as cases of L-bifunctional protein deficiency probably should be reexamined for a possible d-bifunctional protein deficiency.

Cloning and expression of cDNA for a newly identified isozyme of bovine liver 3-hydroxyacyl-CoA dehydrogenase and its import into mitochondria.

cDNA for a heretofore undescribed mitochondrial 3-hydroxyacyl-CoA dehydrogenase, designated as the type II enzyme with different molecular and catalytic properties, compared to those of the classical mitochondrial beta-oxidation enzyme (type I enzyme), was cloned from a bovine liver cDNA library. Nucleotide sequence of the cDNA encoded 261 amino acids with a subunit molecular weight of 27,140. The deduced primary structure of the type II enzyme showed no significant homology to the reported amino acid sequence of the classical 3-hydroxyacyl-CoA dehydrogenases. On SDS-PAGE, no differences in subunit molecular weights were observed among the in vitro translation products, the recombinant type II enzyme produced in Escherichia coli and the purified enzyme. NH2-terminal and COOH-terminal amino acid sequence analysis of the purified type II enzyme revealed that the mature enzyme had not been proteolytically processed. The in vitro translation products of the type II enzyme were efficiently incorporated into isolated rat liver mitochondria, without changes in size, thereby suggesting that unlike other mitochondrial enzymes of fatty acid beta-oxidation, the type II enzyme had no cleavable signal peptide upon import into mitochondria.

Interaction of LDL receptor-related protein 4 (LRP4) with postsynaptic scaffold proteins via its C-terminal PDZ domain-binding motif, and its regulation by Ca2+/calmodulin-dependent protein kinase II

We cloned here a full‐length cDNA of Dem26[ Tian et al. (1999)Mol. Brain Res., 72, 147–157], a member of the low‐density lipoprotein (LDL) receptor gene family from the rat brain. We originally named the corresponding protein synaptic LDL receptor‐related protein (synLRP) [ Tian et al. (2002) Soc. Neurosci. Abstr., 28, 405] and have renamed it LRP4 to accord it systematic nomenclature (GenBankTM accession no. AB073317). LRP4 protein interacted with postsynaptic scaffold proteins such as postsynaptic density (PSD)‐95 via its C‐terminal tail sequence, and associated with N‐methyl‐d‐aspartate (NMDA)‐type glutamate receptor subunit. The mRNA of LRP4 was localized to dendrites, as well as somas, of neuronal cells, and the full‐length protein of 250 kDa was highly concentrated in the brain and localized to various subcellular compartments in the brain, including synaptic fractions. Immunocytochemical study using cultured cortical neurons suggested surface localization in the neuronal cells both in somas and dendrites. Ca2+/calmodulin‐dependent protein kinase II (CaMKII) phosphorylated the C‐terminal cytoplasmic region of LRP4 at Ser1887 and Ser1900, and the phosphorylation at the latter site suppressed the interaction of the protein with PSD‐95 and synapse‐associated protein 97 (SAP97). These findings suggest a postsynaptic role for LRP4, a putative endocytic multiligand receptor, and a mechanism in which CaMKII regulates PDZ‐dependent protein–protein interactions and receptor dynamics.

Turnover of enzymes of peroxisomal β-oxidation in rat liver

Male Wistar rats were given a diet containing 2% (w/w) di-ethylhexyl)-phthalate (DEHP), a peroxisomal proliferator, for 4 weeks. The activities of enzymes of peroxisomal beta-oxidation and of catalase were markedly increased by the DEHP administration. The time required to reach halfway to the maximal induction for enzymes of peroxisomal beta-oxidation was 5--7 days, whereas that for catalase was 3 days. A separate DEHP group was placed on the control diet after 14 days of feeding with the DEHP diet. On the withdrawal of DEHP, activities of enzymes of the beta-oxidation system and of catalase decreased to the control levels with a half-life of 2--3 days. Responses of some mitochondrial enzymes involved in fatty acid oxidation are also described.

Association of Membrane Rafts and Postsynaptic Density: Proteomics, Biochemical, and Ultrastructural Analyses

J. Neurochem. (2011) 119, 64–77.

Brain-specific potential guanine nucleotide exchange factor for Arf, synArfGEF (Po), is localized to postsynaptic density

We cloned from a rat brain cDNA library a novel cDNA and named it a potential synaptic guanine nucleotide exchange factor (GEF) for Arf (synArfGEF (Po)) (GenBank Accession no. AB057643) based on its domain structure and localization. The cloned gene was 7410 bases long with a 3585‐bp coding sequence encoding a protein of 1194 amino acids. The deduced protein contained a coiled‐coil structure in the N‐terminal portion followed by Sec7 and Plekstrin homology (PH) domains. Thus, the protein was a member of the Sec7 family of proteins, GEFs. Conservation of the ADP‐ribosylation factor (Arf)‐binding sequence suggested that the protein was a GEF for Arf. The gene was expressed specifically in the brain, where it exhibited region‐specific expression. The protein was highly enriched in the postsynaptic density (PSD) fraction prepared from the rat forebrain. Uniquely, the protein interacted with PSD‐95, SAP97 and Homer/Vesl 1/PSD‐Zip45 via its C‐terminal PDZ‐binding motif and co‐localized with these proteins in cultured cortical neurons. These results supported its localization in the PSD. The postsynaptic localization was also supported by immunohistochemical examination of the rat brain. The mRNA for the synArfGEF was also localized to dendrites, as well as somas, of neuronal cells. Thus, both the mRNA and the protein were localized in the postsynaptic compartments. These results suggest a postsynaptic role of synArfGEF in the brain.

Riboflavin deficiency and β-oxidation systems in rat liver

Weanling rats were fed a riboflavin-deficient diet. The mitochondrial fatty acid oxidation in liver was depressed in riboflavin deficiency but restored after supplementation of riboflavin. Among the enzymes involved in this system, only the acyl-CoA dehydrogenase (EC 1.3.99.2 and 1.3.99.3) activities varied with the change in fatty acid oxidation. An accumulation of the apoforms of acyl-CoA dehydrogenases was found in riboflavin deficiency. The levels of electron transfer flavoprotein and other enzymes involved in the β-oxidation system remained unchanged. The peroxisomal fatty acid oxidation and levels of individual enzymes of this system remained constant. No accumulation of the apoform of acyl-CoA oxidase was observed under simple, riboflavin-deficient conditions. However, accumulation of a large amount of apo-acyl-CoA oxidase was observed when the peroxisomal system was induced by administration of a peroxisome proliferator, di(2-ethylhexyl)phthalate, under riboflavin-deficient conditions.

Induction of peroxisomal β-oxidation by the administration of acetylsalicylic acid

Abstract The effects of acetylsalicylic acid (ASA) on the fatty acid oxidation system of liver peroxisomes were examined. Male Wistar rats were fed powdered diet containing 1% ( w w ) ASA for 10 days. Peroxisomal palmitoyl-CoA oxidation of rat liver, which is cyanide insensitive, increased by sevenfold after the administration of ASA. Activities of component enzymes of this system were also increased. The effects of salicylic acid (SA) on the induction of peroxisomal β-oxidation were nearly the same as those of ASA. p -Aminosalicylic acid (PAS) and gentisic acid (GA), however, did not cause the alteration of this system. The activity-time curve of the peroxisomal β-oxidation system was studied. The time to reach half-way to maximal induction by the administration of ASA was 3.5 days and the half-life of the system, estimated by drug withdrawal, was 2.0 days. A dose-dependent increase in activity of the system was observed. The results demonstrate that ASA results in proliferation of a peroxisomal fatty acyl-CoA oxidation system in the rat liver, and that the effects of ASA on this system are nearly the same as those of other peroxisomal proliferators which are unrelated in the chemical structures.