Cholesteryl ester levels are elevated in the caudate and putamen of Huntington's disease patients.

Huntington's disease (HD) is an autosomal dominant neurodegenerative illness caused by a mutation in the huntingtin gene (HTT) and subsequent protein (mhtt), to which the brain shows a region-specific vulnerability. Disturbances in neural cholesterol metabolism are established in HD human, murine and cell studies; however, cholesteryl esters (CE), which store and transport cholesterol in the brain, have not been investigated in human studies. This study aimed to identify region-specific alterations in the concentrations of CE in HD. The Victorian Brain Bank provided post-mortem tissue from 13 HD subjects and 13 age and sex-matched controls. Lipids were extracted from the caudate, putamen and cerebellum, and CE were quantified using targeted mass spectrometry. ACAT 1 protein expression was measured by western blot. CE concentrations were elevated in HD caudate and putamen compared to controls, with the elevation more pronounced in the caudate. No differences in the expression of ACAT1 were identified in the striatum. No remarkable differences in CE were detected in HD cerebellum. The striatal region-specific differences in CE profiles indicate functional subareas of lipid disturbance in HD. The increased CE concentration may have been induced as a compensatory mechanism to reduce cholesterol accumulation.

Dictyostelium acetoacetyl-CoA thiolase is a dual-localizing enzyme that localizes to peroxisomes, mitochondria and the cytosol.

Acetoacetyl-CoA thiolase is an enzyme that catalyses both the CoA-dependent thiolytic cleavage of acetoacetyl-CoA and the reverse condensation reaction. In Dictyostelium discoideum, acetoacetyl-CoA thiolase (DdAcat) is encoded by a single acat gene. The aim of this study was to assess the localization of DdAcat and to determine the mechanism of its cellular localization. Subcellular localization of DdAcat was investigated using a fusion protein with GFP, and it was found to be localized to peroxisomes. The findings showed that the targeting signal of DdAcat to peroxisomes is a unique nonapeptide sequence (15RMYTTAKNL23) similar to the conserved peroxisomal targeting signal-2 (PTS-2). Cell fractionation experiments revealed that DdAcat also exists in the cytosol. Distribution to the cytosol was caused by translational initiation from the second Met codon at position 16. The first 18 N-terminal residues also exhibited function as a mitochondrial targeting signal (MTS). These results indicate that DdAcat is a dual-localizing enzyme that localizes to peroxisomes, mitochondria and the cytosol using both PTS-2 and MTS signals, which overlap each other near the N-terminus, and the alternative utilization of start codons.

Assessing the function of mitochondria-associated ER membranes.

Mitochondria-associated membranes or MAMs are specific regions within the endoplasmic reticulum in close apposition to mitochondria. These contacts between both organelles are involved in the regulation of several cellular functions such as the import of phosphatidylserine into mitochondria from the ER for decarboxylation to phosphatidylethanolamine, cholesterol esterification, calcium signaling, mitochondrial shape and motility, autophagy, and apoptosis. Recently, MAM alterations have been described to underlie some neurodegenerative diseases, including Alzheimer's disease. In this chapter, we describe and discuss some of the methods to isolate and assay this interesting cellular region.

Taxol biosynthesis: Identification and characterization of two acetyl CoA:taxoid-O-acetyl transferases that divert pathway flux away from Taxol production.

Two cDNAs encoding taxoid-O-acetyl transferases (TAX 9 and TAX 14) were obtained from a previously isolated family of Taxus acyl/aroyl transferase cDNA clones. The recombinant enzymes catalyze the acetylation of taxadien-5alpha,13alpha-diacetoxy-9alpha,10beta-diol to generate taxadien-5alpha,10beta,13alpha-tri-acetoxy-9alpha-ol and taxadien-5alpha,9alpha,13alpha-triacetoxy-10beta-ol, respectively, both of which then serve as substrates for a final acetylation step to yield taxusin, a prominent side-route metabolite of Taxus. Neither enzyme acetylate the 5alpha- or the 13alpha-hydroxyls of taxoid polyols, indicating that prior acylations is required for efficient peracetylation to taxusin. Both enzymes were kinetically characterized, and the regioselectivity of acetylation was shown to vary with pH. Sequence comparison with other taxoid acyl transferases confirmed that primary structure of this enzyme type reveals little about function in taxoid metabolism. Unlike previously identified acetyl transferases involved in Taxol production, these two enzymes appear to act exclusively on partially acetylated taxoid polyols to divert the Taxol pathway to side-route metabolites.

Practical assay method of cytosolic acetoacetyl-CoA thiolase by rapid release of cytosolic enzymes from cultured lymphocytes using digitonin.

We designed a simple approach to determine cytosolic acetoacetyl-CoA thiolase (CT) activity for differential diagnosis of ketone body catabolic defects, using rapid cell-subfractionation of cultured lymphocytes with digitonin. Efficiency of cell subfractionation was determined by measurement of lactate dehydrogenase and citrate synthetase as marker enzymes for cytosol and organelle fractions, respectively, and confirmed by immunotitration and immunoblotting using antibodies against cytosolic and mitochondrial thiolases, respectively. In the condition of best separation taken in the presence of 1 mg/ml digitonin, acetoacetyl-CoA thiolase activities in the presence of K+ ion in the cytosol and organelle fractions were 138.3+/-39.2 and 84.0+/-16.2 nmol/min/ml, respectively. The thiolase activity in the organelle fraction was doubled by the presence of K+ ion, whereas that in the cytosol fraction was not affected. The thiolase activity in the organelle fraction was reduced by the treatment of anti-mitochondrial acetoacetyl-CoA thiolase (T2) antibody but not by anti-CT antibody. On the other hand, that in the cytosol fraction was significantly decreased by anti-CT antibody but not by anti-T2 antibody. These data suggested that T2 was collected in the organelle fraction, and that CT activity could be assessed by measurement of the thiolase activity in the cytosolic fraction. Succinyl-CoA: 3-ketoacid CoA transferase (SCOT), whose defect is the third inherited disorder of ketone body catabolism, was collected in the organelle fraction. Hence, this method will prove to be useful for accurate assessment of defects of CT as well as T2 or SCOT, all involved in ketone body catabolism.

Effect of centrifugation at 2G for 14 days on metabolic enzymes of the tibialis anterior and soleus muscles.

BACKGROUND: The enzyme composition of different muscle types vary greatly, leading to different changes of enzyme level caused by exposure to various stimuli. METHODS: Male Wistar rats were centrifuged at 2G in a 12-ft radius centrifuge for 14 d. Tibialis anterior (TA) and soleus muscles from four centrifuge and four control rats were analyzed for three enzymes characteristic of fast twitch muscles (phosphofructokinase, glycerol-3-phosphate dehydrogenase, and pyruvate kinase), and four enzymes characteristic of slow twitch muscles (hexokinase, mitochondrial thiolase, B-hydroxyacyl CoA dehydrogenase, and citrate synthase). RESULTS: The centrifuged TA muscles lost 15% of their weight; the corresponding soleus muscles gained 4%. Calculated on the basis of dry weight, the fast twitch enzyme activities were reduced 3-15% in the TA muscles but increased 10-23% in the soleus muscles. The slow twitch enzymes were reduced 18-30% in TA muscles but were almost unchanged in the soleus muscles. When calculated on the basis of total muscle weight, all of the enzymes in TA muscles were significantly reduced by centrifugation. In contrast, in soleus muscles, on the basis of total muscle weight, centrifugation caused an average increase of 22% in the fast twitch enzymes but only marginal changes in the slow twitch enzymes.

Tissue-specific distribution of a peroxisomal 46-kDa protein related to the 58-kDa protein (sterol carrier protein x; sterol carrier protein 2/3-oxoacyl-CoA thiolase).

The complete sequence of the nonspecific lipid-transfer protein (nsL-TP; sterol carrier protein 2) including the presequence is present at the C-terminus (residues 405-547) of a 58-kDa protein. To be able to study this 58-kDa protein without the interference of nsL-TP, antibodies were raised against predicted epitope regions in the N-terminal part (peptide I, residues 23-43; peptide II, residues 130-149). Using these antibodies, rat tissues were analyzed by immunoblotting. In rat liver, in addition to the 58-kDa protein the antibody against peptide I (alpha-58K23) as well as the antibody against peptide II (alpha-58K130) detected a 46-kDa protein. This suggests that both peptide sequences are present in this 46-kDa protein. Both the 46- and the 58-kDa-proteins were abundantly present in liver and adrenals, but could also be detected in brain, kidney, heart, lung, testes, and ovary. This distribution was observed in tissues from both male and female rats. Immunogold labeling of cryosections of liver showed that alpha-58K23 labels the peroxisomes. From double-labeling experiments using alpha-nsL-TP and alpha-58K23 we conclude that the 46-kDa protein is peroxisomal. We propose that in the peroxisomes the protease that processes pre-nsL-TP also cleaves the 58-kDa protein giving rise to the 46-kDa protein and nsL-TP. In addition to the 58- and 46-kDa proteins, an immunoreactive 44-kDa protein was prominently present in rat heart and at low levels also in small intestine and brain. Immunogold labeling of cryosections of heart and Western blotting of purified mitochondria showed that the 44-kDa protein is localized in the mitochondria. The 44-kDa protein was shown to be identical to mitochondrial sarcomeric creatine kinase, which has a peptide segment of five amino acid residues in common with peptide I.

The SH3 domain of the Saccharomyces cerevisiae peroxisomal membrane protein Pex13p functions as a docking site for Pex5p, a mobile receptor for the import PTS1-containing proteins.

We identified a Saccharomyces cerevisiae peroxisomal membrane protein, Pex13p, that is essential for protein import. A point mutation in the COOH-terminal Src homology 3 (SH3) domain of Pex13p inactivated the protein but did not affect its membrane targeting. A two-hybrid screen with the SH3 domain of Pex13p identified Pex5p, a receptor for proteins with a type I peroxisomal targeting signal (PTS1), as its ligand. Pex13p SH3 interacted specifically with Pex5p in vitro. We determined, furthermore, that Pex5p was mainly present in the cytosol and only a small fraction was associated with peroxisomes. We therefore propose that Pex13p is a component of the peroxisomal protein import machinery onto which the mobile Pex5p receptor docks for the delivery of the selected PTS1 protein.

Identification of Pex13p a peroxisomal membrane receptor for the PTS1 recognition factor.

We have identified an S. cerevisiae integral peroxisomal membrane protein of M of 42,705 (Pex13p) that is a component of the peroxisomal protein import apparatus. Pex13p's most striking feature is an src homology 3 (SH3) domain that interacts directly with yeast Pex5p (former Pas10p), the recognition factor for the COOH-terminal tripeptide signal sequence (PTS1), but not with Pex7p (former Pas7p), the recognition factor for the NH2-terminal nonapeptide signal (PTS2) of peroxisomal matrix proteins. Hence, Pex13p serves as peroxisomal membrane receptor for at least one of the two peroxisomal signal recognition factors. Cells deficient in Pex13p are unable to import peroxisomal matrix proteins containing PTS1 and, surprisingly, also those containing PTS2. Pex13p deficient cells retain membranes containing the peroxisomal membrane protein Pex11p (former Pmp27p), consistent with the existence of independent pathways for the integration of peroxisomal membrane proteins and for the translocation of peroxisomal matrix proteins.

Prenatal diagnosis of succinyl-coenzyme A:3-ketoacid coenzyme A transferase deficiency.

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency is a rare disorder of ketone body catabolism. In the present study, we prenatally diagnosed SCOT deficiency in a fetus in a family of which the proband was the first patient with SCOT deficiency identified in Japan, by analysis of enzyme activity levels in samples of chorionic villi and cultured amniocytes. In the fetus of the family, SCOT activity was not detected in either chorionic villi or cultured amniocytes. Since the levels of SCOT activity in control chorionic villi were close to our minimal detectable level and were much lower than those in control cultured amniocytes, enzyme assay in cultured amniocytes was more feasible than that in chorionic villi for prenatal diagnosis of SCOT deficiency. No elevated accumulation of 3-hydroxybutyrate or acetoacetate was detected in the amniotic fluid of the fetus. To our knowledge, this report is the first of prenatal diagnosis of SCOT deficiency.

Localization of peroxisomal 3-oxoacyl-CoA thiolase in particles of varied density in rat liver: implications for peroxisome biogenesis.

In this paper we report on the subcellular localization of peroxisomal thiolase in rat liver using density-gradient centrifugation and immunoelectron microscopy. The results obtained show that peroxisomes display great biochemical heterogeneity and can not be regarded as one homogeneous population of particles. We conclude that rat liver contains at least three distinct populations of peroxisomes, which are present both in normal-fed rats as well in rats treated with a plasticizer, di-(2-ethylhexyl)phthalate, known to induce peroxisomes. The following types of peroxisomes could be discerned: (1) Low-density peroxisomal particles containing 69-kDa peroxisomal membrane protein (PMP), dihydroxyacetonephosphate acyltransferase (DHAPAT) and the precursor form of peroxisomal thiolase (44-kDa). (2) Intermediate-density peroxisomal particles containing 69-kDa peroxisomal membrane protein, dihydroxyacetonephosphate acyltransferase, both 41-kDa (mature) and 44-kDa (immature) peroxisomal thiolase, catalase and D-aminoacid oxidase. (3) High-density peroxisomes containing 69-kDa peroxisomal membrane protein, dihydroxyacetonephosphate acyltransferase, 41-kDa thiolase, catalase and D-aminoacid oxidase.

Purification of an AU-rich RNA binding protein from Sarcophaga peregrina (flesh fly) and its identification as a Thiolase.

A protein that binds to the AU-rich sequence in the 3'-untranslated region of sarcotoxin IIA mRNA was purified from a Sarcophaga pupal extract to near homogeneity. The molecular mass of this protein was estimated to be 39 kDa by SDS-polyacrylamide gel electrophoresis. The partial amino acid sequences of two peptides obtained from the 39 kDa protein showed striking similarities to partial amino acid sequences of rat and yeast 3-oxoacyl-CoA thiolase, suggesting that this protein is a Sarcophaga thiolase. In fact, the purified 39 kDa protein was found to have thiolase activity. Moreover, rat mitochondrial 3-oxoacyl-CoA thiolase showed affinity to the AU-rich RNA. These suggest that the RNA binding activity is an intrinsic character of thiolase.

Biochemical and immunochemical study of seven families with 3-ketothiolase deficiency: diagnosis of heterozygotes using immunochemical determination of the ratio of mitochondrial acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase proteins.

The possibility of identifying heterozygotes of 3-ketothiolase deficiency, an inborn error of metabolism caused by a defect of mitochondrial acetoacetyl-CoA thiolase (T2), was tested in seven unrelated families by using enzymatic assay of thiolase activity and immunoblot analysis. The ratio of acetoacetyl-CoA thiolase activities, in the presence and absence of K+ ion (+K/-K ratio), in fibroblasts from 15 normal controls was around 2.0 (1.8 to 2.4), whereas the +K/-K ratio in eight patients was always 1.0. The ratio for the 13 obligate carriers ranged from 1.4 to 1.9, causing a minor overlap with control. Identification of heterozygote cells by immunoblot analysis, using anti-T2 antibody alone as a probe, was difficult, as previously reported. We therefore carried out immunoblot analysis, using as probes a mixture of anti-T2 antibody and the antibody against mitochondrial 3-ketoacyl-CoA thiolase (T1), another mitochondrial thiolase, and determined the ratio of the intensities of the T2 and T1 bands (T2/T1 ratio) using a densitometer. When the T2/T1 ratio was calculated, there was no overlap between the heterozygotes and normal controls. Hence, the heterozygotes can be unambiguously identified using this method. The thiolase activities and T2/T1 proteins in immunoblotting were detectable in peripheral lymphocytes, rectal mucosa, amniocytes, and liver. Thus, the postnatal diagnosis of 3-ketothiolase deficiency can be readily made using lymphocytes or rectal mucosa. The applicability of these methods in amniocytes indicates that prenatal diagnosis of this disease should be possible.

Morphometry of peroxisomes and immunolocalization of peroxisomal proteins in the liver of patients with generalised peroxisomal disorders.

Hepatic peroxisomes were studied by morphometric and immunocytochemical techniques in control patients and in four Zellweger syndrome patients, two infantile Refsum's (IRD) patients, one neonatal adrenoleukodystrophy (NALD) patient, and three patients with peroxisomal disorders (PD) which do not fit any currently recognised classification, but have disorders involving a defect in peroxisomal biogenesis. Peroxisomes which were ultrastructurally abnormal and greatly reduced in size and/or number were found in two of the Zellweger syndrome patients, and the NALD and IRD patients. There was variation in their numerical density ranging from none at all in two of the Zellweger syndrome patients to normal numbers in the IRD patients. In most patients there was a decrease in the immunolabelling of catalase over the peroxisomes. In the Zellweger syndrome and NALD patients, the small, abnormal peroxisomes did not label for any of the beta-oxidation proteins. The IRD patients and the PD patients however, were heterogeneous with respect to beta-oxidation labelling. The ultrastructural heterogeneity of peroxisomes in these peroxisomal disorders patients indicates there may be genotypic differences between the major groups and also within each group. The common factor in all the patients in this study where peroxisomes were present was the presence in the hepatic peroxisomes of an electron dense centre which did not label immunocytochemically for catalase or the beta-oxidation enzymes. This electron dense centre may indicate a structural abnormality in the peroxisomes in these patients.

Investigation of peroxisomal lipid beta-oxidation enzymes in guinea pig liver peroxisomes by immunoblotting and immunocytochemistry.

We investigated the immunoreactivity of the peroxisomal lipid beta-oxidation enzymes acyl-CoA oxidase, trifunctional protein, and thiolase in guinea pig liver and compared it with that of homologous proteins in rat, using immunoblotting of highly purified peroxisomal fractions and monospecific antibodies to rat proteins. In addition, the immunocytochemical localization of beta-oxidation enzymes in guinea pig liver was compared with that of catalase. All antibodies showed crossreactivity between the two species, indicating that these peroxisomal proteins have been well conserved, although all exhibited some differences with respect to molecular size and, in the case of acyl-CoA oxidase, in frequency of the immunoreactive bands. In the latter case, a distinct second band in the 70 KD range was observed in guinea pig, in addition to the regular band due to subunit A present in rat liver. This novel band could be due either to trihydroxycoprostanoyl-CoA oxidase or to the non-inducible branched chain fatty acid oxidase described recently. All three beta-oxidation enzymes were immunolocalized by light and electron microscopy to the matrix of peroxisomes, in contrast to catalase, which is also found in the cytoplasm and the nucleus of hepatocytes in guinea pig liver.

Immunoelectron microscopic localization of thiolases, beta-oxidation enzymes of an n-alkane-utilizable yeast, Candida tropicalis.

The location of acetoacetyl-CoA thiolase (T-I) and 3-ketoacyl-CoA thiolase (T-III), enzymes of the fatty acid beta-oxidation system, was studied in n-alkane-grown Candida tropicalis cells by immunoelectron microscopy using a post-embedding method with colloidal gold conjugated IgG. The deposition of gold particles for T-I was detected in the microbodies and cytoplasm and that of gold particles for T-III specifically in the microbodies. The double labeling technique confirmed that T-I and T-III occurred concurrently in a microbody and T-I also in cytoplasm. These results were consistent with the biochemical data based on subcellular fractionation and indicated that the yeast beta-oxidation system operates efficiently only in the microbodies.

Presence of acetyl-transferases and adenylyl-transferases in gentamicin-resistant transconjugants.

UNLABELLED: The aim of this work was to test the production of aminoglycoside modifying enzymes in 20 gentamicin-resistant transconjugants obtained from clinical strains of Entero-bacteriaceae. The susceptibility to aminoglycosides was determined by disc diffusion method and agar dilution method according to European Committee for Clinical Laboratory Standards, 1988. The transfer of gentamicin-resistance R-plasmids was made by conjugation on a solid medium with recipients E. coli K12. Phosphocellulose paper binding assay with 14C acetyl. CoA and 14C.ATP by Haas and Dowding was performed to reveal the enzyme production. Four different acetyl-transferases have been found: AAC/3/I, AAC/3/-V, AAC/3/-IV which modify gentamicin, and AAC/6'/-I with activity on amikacin. Only two of the transconjugants showed adenylyl-transferase activity:AAD/2"/. Some of strains tested possessed two enzymes. The most interesting finding was that the majority of strains owned AAC/3/-IV, which modifies apramycin. This was be explained with the fact that apramycin is still in a large use for animal husbandry in Bulgaria. IN CONCLUSION: four different acetyl-transferases: AAC/3/-I, AAC/3/-V, AAC/3/-IV and AAC/6'/-I and the adenylyl-transferase AAD/2"/ were found to be the biochemical mechanisms of resistance to aminoglycosides in 20 gentamicin-resistant transconjugants.

Phenotypic heterogeneity in cultured skin fibroblasts from patients with disorders of peroxisome biogenesis belonging to the same complementation group.

We have studied fibroblast cell lines derived from a control subject (cell line 85AD5035F) and three patients clinically described as having the Zellweger syndrome (cell line W78/515), the infantile form of Refsum disease (cell line BOV84AD) and hyperpipecolic acidaemia (cell line GM3605), respectively. The mutant cell lines belonged to the same complementation group. The fibroblasts were cultured under identical conditions and were harvested at different time intervals after reaching confluence. Several peroxisomal parameters were determined. In agreement with previous reports, a lowered enzymic activity of acyl-CoA: dihydroxyacetonephosphate acyltransferase and a decrease in latent catalase clearly distinguished the patient cell lines from the control cell line. However, the cell lines exhibited a phenotypic heterogeneity. This was most strikingly encountered when cells were processed for indirect immunofluorescence microscopy and stained with anti-(catalase). The control cells exhibited a punctate fluorescence, which is indicative of the presence of catalase in peroxisomes. In the mutant cell line W78/515 a diffuse fluorescence was observed, indicative of the presence of catalase in the cytosol. In the other two mutant cell lines a punctate fluorescence was observed in some of the cells. Moreover, clear differences in the extent of proteolytic processing of acyl-CoA oxidase were detected. The mutant cell line BOV84AD displayed a control-like pattern with all molecular forms of acyl-CoA oxidase (72, 52 and 20 kDa) present, whereas in the W78/515 cell line only the 72 kDa component could be visualised. The GM3605 cell line was intermediate in this respect.

Identification of a catalase-negative sub-population of peroxisomes induced in mouse liver by clofibrate.

The peroxisomal compartment in mouse liver was investigated using rate sedimentation of liver subfractions on sucrose density gradients. Treatment of mice with clofibrate, a hypolipidemic agent and peroxisome proliferator, resulted in the formation of small particles which were devoid of catalase and urate oxidase, but which were identified as peroxisomal on the basis of content of the clofibrate-induced peroxisomal beta-oxidation enzymes (fatty acyl-CoA oxidase, hydratase/dehydrogenase bifunctional protein, and thiolase) and the 68 kDa peroxisomal integral membrane protein. Immunoelectron microscopy confirmed the membrane-bound organellar nature and enzyme composition of these particles. These particles were absent in normal mice, and were increased to a maximal level within 2 days of clofibrate treatment. These data have been taken as indicative of a role of these particles in the mechanism of drug-induced peroxisome proliferation.