Crystal structure of human AUH protein, a single-stranded RNA binding homolog of enoyl-CoA hydratase.

BACKGROUND: The AU binding homolog of enoyl-CoA hydratase (AUH) is a bifunctional protein that has two distinct activities: AUH binds to RNA and weakly catalyzes the hydration of 2-trans-enoyl-coenzyme A (enoyl-CoA). AUH has no sequence similarity with other known RNA binding proteins, but it has considerable sequence similarity with enoyl-CoA hydratase. A segment of AUH, named the R peptide, binds to RNA. However, the mechanism of the RNA binding activity of AUH remains to be elucidated. RESULTS: We determined the crystal structure of human AUH at 2.2 A resolution. AUH adopts the typical fold of the enoyl-CoA hydratase/isomerase superfamily and forms a hexamer as a dimer of trimers. Interestingly, the surface of the AUH hexamer is positively charged, in striking contrast to the negatively charged surfaces of the other members of the superfamily. Furthermore, wide clefts are uniquely formed between the two trimers of AUH and are highly positively charged with the Lys residues in alpha helix H1, which is located on the edge of the cleft and contains the majority of the R peptide. A mutational analysis showed that the lysine residues in alpha helix H1 are essential to the RNA binding activity of AUH. CONCLUSIONS: Alpha helix H1 exposes a row of Lys residues on the solvent-accessible surface. These characteristic Lys residues are named the "lysine comb." The distances between these Lys residues are similar to those between the RNA phosphate groups, suggesting that the lysine comb may continuously bind to a single-stranded RNA. The clefts between the trimers may provide spaces sufficient to accommodate the RNA bases.     

AU-rich RNA-binding induces changes in the quaternary structure of AUH

The human AU RNA binding protein/enoyl-Coenzyme A hydratase (AUH) is a 3-hydroxy-3-methylglutaconyl-CoA dehydratase in the leucine degradation pathway. It also possesses an RNA-binding activity to AUUU repeats, which involves no known conserved RNA-binding domains and is seemingly unrelated to the enzymatic activity. In this study, we performed mass spectrometric analyses to elucidate the oligomeric states of AUH in the presence and absence of RNA. With a short RNA (AUUU) or without RNA, AUH mainly exists as a trimer in solution. On the other hand, the AUH trimer dimerizes upon binding to one molecule of a long RNA containing 24 repeats of the AUUU motif, (AUUU)(24)A. AUH was crystallized with the long RNA. Although the RNA was disordered in the crystalline lattice, the AUH structure was determined as an asymmetric dimer of trimers with a kink in the alignment of the trimer axes, resulting in the formation of two clefts with significantly different sizes.     

Relationship between Enoyl-CoA Hydratase and a Peroxisomal Bifunctional Enzyme,Enoyl-CoA Hydratase/3-Hydroxyacyl- CoA Dehydrogenase,from an n-Alkane-utilizing Yeast,Candida tropicalis

A protein exhibiting only enoyl-CoA hydratase (EC 4.2.1.17) activity was purified from an n- alkane-grown yeast, Candida tropicalis. This enzyme had a homotetrameric form composed of subunits with a molecular mass of 36kDa. On the other hand, a bifunctional enzyme exhibiting enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) activities was obtained from the same yeast cells when purified in the presence of protease inhibitors, phenylmethylsulfonyl fluoride, antipain and chymostatin. The enzyme had a molecular mass of 105 kDa and was a monomeric form. Limited proteolysis of the bifunctional enzyme with α-chymotrypsin yielded a peptide mixture containing a 36 kDa fragment, the mixture showing about 76% of the original enoyl-CoA hydratase activity but no 3-hydroxyacyl-CoA dehydrogenase activity. Comparison of the peptide maps of the purified enoyl-CoA hydratase and the 36 kDa fragment obtained from the bifunctional enzyme showed the similarity of these proteins. These results strongly suggest that the domain of enoyl-CoA hydratase is separable from the bifunctional enzyme through the action of a certain protease.     

3-Methylglutaconic aciduria type I is caused by mutations in AUH

3-Methylglutaconic aciduria type I is an autosomal recessive disorder clinically characterized by various symptoms ranging from delayed speech development to severe neurological handicap. This disorder is caused by a deficiency of 3-methylglutaconyl-CoA hydratase, one of the key enzymes of leucine degradation. This results in elevated urinary levels of 3-methylglutaconic acid, 3-methylglutaric acid, and 3-hydroxyisovaleric acid. By heterologous expression in Escherichia coli, we show that 3-methylglutaconyl-CoA hydratase is encoded by the AUH gene, whose product had been reported elsewhere as an AU-specific RNA-binding protein. Mutation analysis of AUH in two patients revealed a nonsense mutation (R197X) and a splice-site mutation (IVS8-1G-->A), demonstrating that mutations in AUH cause 3-methylglutaconic aciduria type I.     

Novel fatty acid beta-oxidation enzymes in rat liver mitochondria. II. Purification and properties of enoyl-coenzyme A (CoA) hydratase/3-hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase trifunctional protein.

Long-chain 3-hydroxyacyl-CoA dehydrogenase was extracted from the washed membrane fraction of frozen rat liver mitochondria with buffer containing detergent and then was purified. This enzyme is an oligomer with a molecular mass of 460 kDa and consisted of 4 mol of large polypeptide (79 kDa) and 4 mol of small polypeptides (51 and 49 kDa). The purified enzyme preparation was concluded to be free from the following enzymes based on marked differences in behavior of the enzyme during purification, molecular masses of the native enzyme and subunits, and immunochemical properties: enoyl-CoA hydratase, short-chain 3-hydroxyacyl-CoA dehydrogenase, peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein, and mitochondrial and peroxisomal 3-ketoacyl-CoA thiolases. The purified enzyme exhibited activities toward enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase together with the long-chain 3-hydroxyacyl-CoA dehydrogenase activity. The carbon chain length specificities of these three activities of this enzyme differed from those of the other enzymes. Therefore, it is concluded that this enzyme is not long-chain 3-hydroxyacyl-CoA dehydrogenase; rather, it is enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase trifunctional protein.     

Evidence that the enoyl-CoA hydratase bifunctional protein of mouse liver peroxisomes is identical with the 70,000 dalton peroxisomal membrane protein

Peroxisomal enoyl-CoA hydratase was purified from livers of mice treated with di-(2-ethylhexyl)phthalate and its properties compared with those of the 70 kDa protein present in the membranes prepared by carbonate extraction of peroxisomes. The two proteins had identical subunit molecular masses, of about 70,000 daltons. Limited proteolysis of these proteins using the V8 proteinase of S. aureus yielded identical peptide maps, with these peptides crossreacting with antiserum raised against the 70 kDa membrane protein. These data are consistent with the proposal that the peroxisomal 70 kDa membrane protein and the peroxisomal enoyl-CoA hydratase are the same protein.     

Biochemical characterization of human 3-methylglutaconyl-CoA hydratase and its role in leucine metabolism

The metabolic disease 3-methylglutaconic aciduria type I (MGA1) is characterized by an abnormal organic acid profile in which there is excessive urinary excretion of 3-methylglutaconic acid, 3-methylglutaric acid and 3-hydroxyisovaleric acid. Affected individuals display variable clinical manifestations ranging from mildly delayed speech development to severe psychomotor retardation with neurological handicap. MGA1 is caused by reduced or absent 3-methylglutaconyl-coenzyme A (3-MG-CoA) hydratase activity within the leucine degradation pathway. The human AUH gene has been reported to encode for a bifunctional enzyme with both RNA-binding and enoyl-CoA-hydratase activity. In addition, it was shown that mutations in the AUH gene are linked to MGA1. Here we present kinetic data of the purified gene product of AUH using different CoA-substrates. The best substrates were (E)-3-MG-CoA (V(max) = 3.9 U.mg(-1), K(m) = 8.3 microM, k(cat) = 5.1 s(-1)) and (E)-glutaconyl-CoA (V(max) = 1.1 U.mg(-1), K(m) = 2.4 microM, k(cat) = 1.4 s(-1)) giving strong evidence that the AUH gene encodes for the major human 3-MG-CoA hydratase in leucine degradation. Based on these results, a new assay for AUH activity in fibroblast homogenates was developed. The only missense mutation found in MGA1 phenotypes, c.719C>T, leading to the amino acid exchange A240V, produces an enzyme with only 9% of the wild-type 3-MG-CoA hydratase activity.     

Induction, immunochemical identity and immunofluorescence localization of an 80000-molecular-weight peroxisome-proliferation-associated polypeptide (polypeptide PPA-80) and peroxisomal enoyl-CoA hydratase of mouse liver and renal cortex

The hypolipidaemic drugs methyl clofenapate, BR-931, Wy-14643 and procetofen induced a marked proliferation of peroxisomes in the parenchymal cells of liver and the proximal-convoluted-tubular epithelium of mouse kidney. The proliferation of peroxisomes was associated with 6–12-fold increase in the peroxisomal palmitoyl-CoA oxidizing capacity of the mouse liver. Enhanced activity of the peroxisomal palmitoyl-CoA oxidation system was also found in the renal-cortical homogenates of hypolipidaemic-drug-treated mice. The activity of enoyl-CoA hydratase in the mouse liver increased 30–50-fold and in the kidney cortex 3–5-fold with hypolipidaemic-drug-induced peroxisome proliferation in these tissues, and over 95% of this induced activity was found to be heat-labile peroxisomal enzyme in both organs. Sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic analysis of large-particle and microsomal fractions obtained from the liver and kidney cortex of mice treated with hypolipidaemic peroxisome proliferators demonstrated a substantial increase in the quantity of an 80000-mol.wt. peroxisome-proliferation-associated polypeptide (polypeptide PPA-80). The heat-labile peroxisomal enoyl-CoA hydratase was purified from the livers of mice treated with the hypolipidaemic drug methyl clofenapate; the antibodies raised against this electrophoretically homogeneous protein yielded a single immunoprecipitin band with purified mouse liver enoyl-CoA hydratase and with liver and kidney cortical extracts of normal and hypolipidaemic-drug-treated mice. These anti-(mouse liver enoyl-CoA hydratase) antibodies also cross-reacted with purified rat liver enoyl-CoA hydratase and with the polypeptide PPA-80 obtained from rat and mouse liver. Immunofluorescence studies with anti-(polypeptide PPA-80) and anti-(peroxisomal enoyl-CoA hydratase) provided visual evidence for the localization and induction of polypeptide PPA-80 and peroxisomal enoyl-CoA hydratase in the liver and kidney respectively of normal and hypolipidaemic-drug-treated mice. In the kidney, the distribution of these two proteins is identical and limited exclusively to the cytoplasm of proximal-convoluted-tubular epithelium. The immunofluorescence studies clearly complement the biochemical and ultrastructural observations of peroxisome induction in the liver and kidney cortex of mice fed on hypolipidaemic drugs. In addition, preliminary ultrastructural studies with the protein-A–gold-complex technique demonstrate that the heat-labile hepatic enoyl-CoA hydratase is localized in the peroxisome matrix.     

Immunochemical identity of peroxisomal enoyl-CoA hydratase with the peroxisome-proliferation -associated 80,000 mol wt polypeptide in rat liver

Peroxisome proliferators, which induce proliferation of hepatic peroxisomes, have been shown previously to cause a marked increase in an 80,000 mol wt polypeptide predominantly in the light mitochondrial and microsomal fractions of liver of rodents. We now present evidence to show that this hepatic peroxisome-proliferation-associated polypeptide, referred to as polypeptide PPA-80, is immunochemically identical with the multifunctional peroxisome protein displaying heat-labile enoyl-CoA hydratase activity. This conclusion is based on the following observations: (a) the purified polypeptide PPA-80 and the heat- labile enoyl-CoA hydratase from livers of rats treated with the peroxisome proliferators Wy-14,643 {[4-chloro-6(2,3-xylidino)-2-pyrimidinylthio]acetic acid} exhibit identical minimum molecular weights of approximately 80,000 on SDS polyacrylamide gel electrophoresis; (b) these two proteins are immunochemically identical on the basis of ouchterlony double diffusion, immunotitration, rocket immunoelectrophoresis, and crossed immunoelectrophoresis analysis; and (c) the immunoprecipitates formed by antibodies to polypeptide PPA-80 when dissociated on a sephadex G-200 column yield enoyl-CoA hydratase activity. Whether the polypeptide PPA-80 exhibits the activity of other enzyme(s) of the peroxisomal β-oxidation system such as fatty acyl-CoA oxidase activity or displays immunochemical identity with such enzymes remains to be determined. The availability of antibodies to polypeptide PPA-80 and enoyl-CoA hydratase facilitated immunofluorescent and immunocytochemical localization of the polypeptide PPA- 80 and enoyl-CoA hydratase in the rat liver. The indirect immunofluorescent studies with these antibodies provided direct visual evidence for the marked induction of polypeptide PPA-80 and enoyl-CoA hydratase in the livers of rats treated with Wy-14,643. The present studies also provide immunocytochemical evidence for the localization of polypeptide PPA- 80 and the heat-labile enoyl-CoA hydratase in the peroxisome, but not in the mitochondria, of hepatic parenchymal cells. These studies, therefore, provide morphological evidence for the existence of fatty acyl-CoA oxidizing system in peroxisomes. An increase of polypeptide PPA-80 on SDS polyacrylamide gel electrophoretic analysis of the subcellular fractions of liver of rodents treated with lipid-lowering drugs should serve as a reliable and sensitive indicator of enhanced peroxisomal β- oxidation system.     

Propionyl-coenzyme A synthase from Chloroflexus aurantiacus, a key enzyme of the 3-hydroxypropionate cycle for autotrophic CO2 fixation

The 3-hydroxypropionate cycle has been proposed as a new autotrophic CO(2) fixation pathway for the phototrophic green non-sulfur eubacterium Chloroflexus aurantiacus and for some chemotrophic archaebacteria. The cycle requires the reductive conversion of the characteristic intermediate 3-hydroxypropionate to propionyl-CoA. The specific activity of the 3-hydroxypropionate-, CoA-, K(+)-, and MgATP-dependent oxidation of NADPH in autotrophically grown cells was 0.09 micromol min(-1) mg(-1) protein, which was 2-fold down-regulated in heterotrophically grown cells. Unexpectedly, a single enzyme catalyzes the entire reaction sequence: 3-hydroxypropionate + MgATP + CoA + NADPH + H(+) --> propionyl-CoA + MgAMP + PP(i) + NADP(+) + H(2)O. The enzyme was purified 30-fold to near homogeneity and has a very large native molecular mass between 500 and 800 kDa, with subunits of about 185 kDa as judged by SDS-PAGE, suggesting a homotrimeric or homotetrameric structure. Upon incubation of this new enzyme, termed propionyl-CoA synthase, with the proteinase trypsin, the NADPH oxidation function of the enzyme was lost, whereas the enzyme still activated 3-hydroxypropionate to its CoA-thioester and dehydrated it to acrylyl-CoA. SDS-PAGE revealed that the subunits of propionyl-CoA synthase had been cleaved once and the N-terminal amino acid sequences of the two trypsin digestion products were determined. Two parts of the gene encoding propionyl-CoA synthase (pcs) were identified on two contigs of an incomplete genome data base of C. aurantiacus, and the sequence of the pcs gene was completed. Propionyl-CoA synthase is a natural fusion protein of 201 kDa consisting of a CoA ligase, an enoyl-CoA hydratase, and an enoyl-CoA reductase, the reductase domain containing the trypsin cleavage site. Similar polyfunctional large enzymes are common in secondary metabolism (e.g. polyketide synthases) but rare in primary metabolism (e.g. eukaryotic type I fatty acid synthase). These results lend strong support to the operation of the proposed pathway in autotrophic CO(2) fixation.     

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.     

Functional classification of protein 3D structures from predicted local interaction sites

A new approach to the functional classification of protein 3D structures is described with application to some examples from structural genomics. This approach is based on functional site prediction with THEMATICS and POOL. THEMATICS employs calculated electrostatic potentials of the query structure. POOL is a machine learning method that utilizes THEMATICS features and has been shown to predict accurate, precise, highly localized interaction sites. Extension to the functional classification of structural genomics proteins is now described. Predicted functionally important residues are structurally aligned with those of proteins with previously characterized biochemical functions. A 3D structure match at the predicted local functional site then serves as a more reliable predictor of biochemical function than an overall structure match. Annotation is confirmed for a structural genomics protein with the ribulose phosphate binding barrel (RPBB) fold. A putative glucoamylase from Bacteroides fragilis (PDB ID 3eu8) is shown to be in fact probably not a glucoamylase. Finally a structural genomics protein from Streptomyces coelicolor annotated as an enoyl-CoA hydratase (PDB ID 3g64) is shown to be misannotated. Its predicted active site does not match the well-characterized enoyl-CoA hydratases of similar structure but rather bears closer resemblance to those of a dehalogenase with similar fold.     

Biochemical and genetic analyses of ferulic acid catabolism in Pseudomonas sp. Strain HR199.

The gene loci fcs, encoding feruloyl coenzyme A (feruloyl-CoA) synthetase, ech, encoding enoyl-CoA hydratase/aldolase, and aat, encoding beta-ketothiolase, which are involved in the catabolism of ferulic acid and eugenol in Pseudomonas sp. strain HR199 (DSM7063), were localized on a DNA region covered by two EcoRI fragments (E230 and E94), which were recently cloned from a Pseudomonas sp. strain HR199 genomic library in the cosmid pVK100. The nucleotide sequences of parts of fragments E230 and E94 were determined, revealing the arrangement of the aforementioned genes. To confirm the function of the structural genes fcs and ech, they were cloned and expressed in Escherichia coli. Recombinant strains harboring both genes were able to transform ferulic acid to vanillin. The feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase activities of the fcs and ech gene products, respectively, were confirmed by photometric assays and by high-pressure liquid chromatography analysis. To prove the essential involvement of the fcs, ech, and aat genes in the catabolism of ferulic acid and eugenol in Pseudomonas sp. strain HR199, these genes were inactivated separately by the insertion of omega elements. The corresponding mutants Pseudomonas sp. strain HRfcsOmegaGm and Pseudomonas sp. strain HRechOmegaKm were not able to grow on ferulic acid or on eugenol, whereas the mutant Pseudomonas sp. strain HRaatOmegaKm exhibited a ferulic acid- and eugenol-positive phenotype like the wild type. In conclusion, the degradation pathway of eugenol via ferulic acid and the necessity of the activation of ferulic acid to the corresponding CoA ester was confirmed. The aat gene product was shown not to be involved in this catabolism, thus excluding a beta-oxidation analogous degradation pathway for ferulic acid. Moreover, the function of the ech gene product as an enoyl-CoA hydratase/aldolase suggests that ferulic acid degradation in Pseudomonas sp. strain HR199 proceeds via a similar pathway to that recently described for Pseudomonas fluorescens AN103.     

Characterization of protein-binding to the spinach chloroplast psbA mRNA 5' untranslated region.

RNA-binding proteins play a major role in regulating mRNA metabolism in chloroplasts. In this work we characterized two proteins, of 43 and 47 kDa, which bind to the spinach psbA mRNA 5' untranslated region (psbA encoding the D1 protein of photosystem II). The 43 kDa protein, which is present in the stroma and in membranes, co-sediments with a complex of 68S. It was purified, and the N-terminal sequence was determined. Upon homology search it was identified as the chloroplast homologue of the Escherichia coli ribosomal protein S1. The 47 kDa protein, which, in contrast with the 43 kDa protein, sediments with a small sedimentation coefficient, is only detected in the stromal fraction. It is soluble in an uncomplexed form. By deletion analysis, an element within the psbA mRNA 5' untranslated region was identified that is necessary but not sufficient for binding of stromal proteins. The 'central protein binding element' ranges from nucleotide -49 to -9 of the psbA mRNA 5' untranslated region. It comprises the Shine-Dalgarno-like GGAG motif and, 7 nucleotides upstream, an endonucleolytic cleavage site involved in psbA mRNA degradation in vitro . The mechanistic impacts of this region in relation to RNA-binding proteins are discussed.     

MFE1, a member of the peroxisomal hydroxyacyl coenzyme A dehydrogenase family,affects fatty acid metabolism necessary for morphogenesis in Dictyostelium spp

Beta-oxidation of long-chain fatty acids and branched-chain fatty acids is carried out in mammalian peroxisomes by a multifunctional enzyme (MFE) or D-bifunctional protein, with separate domains for hydroxyacyl coenzyme A (CoA) dehydrogenase, enoyl-CoA hydratase, and steroid carrier protein SCP2. We have found that Dictyostelium has a gene, mfeA, encoding MFE1 with homology to the hydroxyacyl-CoA dehydrogenase and SCP2 domains. A separate gene, mfeB, encodes MFE2 with homology to the enoyl-CoA hydratase domain. When grown on a diet of bacteria, Dictyostelium cells in which mfeA is disrupted accumulate excess cyclopropane fatty acids and are unable to develop beyond early aggregation. Axenically grown mutant cells, however, developed into normal fruiting bodies composed of spores and stalk cells. Comparative analysis of whole-cell lipid compositions revealed that bacterially grown mutant cells accumulated cyclopropane fatty acids that remained throughout the developmental stages. Such a persistent accumulation was not detected in wild-type cells or axenically grown mutant cells. Bacterial phosphatidylethanolamine that contains abundant cyclopropane fatty acids inhibited the development of even axenically grown mutant cells, while dipalmitoyl phosphatidylethanolamine did not. These results suggest that MFE1 protects the cells from the increase of the harmful xenobiotic fatty acids incorporated from their diets and optimizes cellular lipid composition for proper development. Hence, we propose that this enzyme plays an irreplaceable role in the survival strategy of Dictyostelium cells to form spores for their efficient dispersal in nature.     

Response of chemically induced hepatocytelike cells in hamster pancreas to methyl clofenapate, a peroxisome proliferator

Administration of N-nitrosobis (2-oxopropyl)amine during peak DNA synthesis of regenerating pancreas in hamsters has been shown to induce hepatocytelike cells in pancreas. We now present evidence to demonstrate that such cells respond to methyl clofenapate, a peroxisome proliferator. The response includes a marked proliferation of peroxisomes and enhanced activity of peroxisomal enzymes enoyl-CoA hydratase (8.5- to 13-fold), [1-14C]-palmitoyl-CoA oxidation (2.8- to 3.9-fold), catalase (1.6 to 3.4-fold), and carnitine acetyltransferase (greater than 2,000-fold). Cytochemical localization of catalase by the alkaline 3,3'-diaminobenzidine procedure and immunofluorescence localization of heat-labile enoyl-CoA hydratase showed that these peroxisome-associated enzymes are localized strictly in pancreatic hepatocytelike cells, while adjacent acinar, duct, and islet cells appeared consistently negative. Morphometric analyses of hepatocytelike cells showed a significant increase in the numerical density and an eightfold increase in the volume density of peroxisomes in methyl clofenapate treated animals. These results demonstrate that the hepatocytelike cells are responsible for the observed peroxisomal enzyme activity in pancreas of hamsters and suggest that the derepressed peroxisome specific genes in these cells respond to a peroxisome proliferator as do parenchymal cells in hamster liver.     

Characterization of the HSD17B4 gene: D-specific multifunctional protein 2/17beta-hydroxysteroid dehydrogenase IV.

The HSD17B4 gene codes for a 80 kDa multifunctional enzyme containing three distinct functional domains and is localized in peroxisomes. The N-terminal part exhibits 3-hydroxyacyl-CoA dehydrogenase and 17beta-hydroxysteroid dehydrogenase activity whereas the central part shows enoyl-CoA hydratase activity. The carboxy-terminal part of the protein has sterol-carrier-protein activity. The protein is widely expressed, however in several tissues like brain, uterus and lung its expression is limited to specific cells like Purkinje cells or luminal epithelium. The HSD17B4 gene consist of 24 exons and 23 introns with classical intron-exon junctions spanning more than 100 kbp. The importance of the HSD17B4 protein is stressed by the identification of patients with severe clinical abnormalities due to mutations in the HSD17B4 gene. We have now checked the consequences of one frequent mutation, G16 S, which results in inactivation of the enzyme due to loss of interaction with NAD+.     

CIRP2, a major cytoplasmic RNA-binding protein in Xenopus oocytes

In an attempt to isolate mRNA-binding proteins we fractionated Xenopus oocyte lysate by oligo(dT)–cellulose chromatography. A 20 kDa protein was the major component of the eluate. cDNA cloning revealed that this protein is a Xenopus homolog of the cold-inducible RNA-binding protein (CIRP) which was originally identified in mammalian cells as a protein that is overexpressed upon a temperature downshift. This Xenopus protein, termed here xCIRP2, is highly expressed in ovary, testis and brain in adult Xenopus tissues. In oocytes it is predominantly localized in the cytoplasm. By biochemical fractionation we provide evidence that xCIRP2 is associated with ribosomes, suggesting that it participates in translational regulation in oocytes. Microinjection of labeled mRNA into oocytes followed by UV cross-linking of the oocyte lysate led to identification of two major RNA-binding activities. Immunoprecipitation of the RNA-binding proteins demonstrated that one is xCIRP2 and that the other contains FRGY2. FRGY2, which is one of the principal constituents of mRNA storage particles involved in translational masking of maternal mRNA, has an RNA-binding domain conserved to those of bacterial cold shock proteins. Possible implications of the highly abundant expression in oocytes of cold shock RNA-binding proteins of both eukaryotic and prokaryotic types are discussed.