Human brain acyl-CoA hydrolase isoforms encoded by a single gene

Acyl-CoA hydrolases are a group of enzymes that catalyze the hydrolysis of acyl-CoA thioesters to free fatty acids and CoA-SH. The human brain acyl-CoA hydrolase (BACH) gene comprises 13 exons, generating several isoforms through the alternative use of exons. Four first exons (1a-1d) can be used, and three patterns of splicing occur at exon X located between exons 7 and 8 that contains an internal 3(')-splice acceptor site and creates premature stop codons. When examined with green fluorescent protein-fusion constructs expressed in Neuro-2a cells, the nuclear localization signal encoded by exon 9 was functional by itself, whereas the whole structure was cytosolic, suggesting nuclear translocation of the enzyme. This was consistent with dual staining of the cytosol and nucleus in certain neurons by immunohistochemistry using anti-BACH antibody. The mitochondrial targeting signals encoded by exons 1b and 1c were also functional and directed mitochondrial localization of BACH isoforms with the signals. Although BACH mRNA containing the sequence derived from exon 1a, but not exon X, was exclusively expressed in human brain, these results suggest that the human BACH gene can express long-chain acyl-CoA hydrolase activity in multiple intracellular compartments by generating BACH isoforms with differential localization signals to affect various cellular functions that involve acyl-CoAs.     

Long-chain acyl-CoA hydrolase in the brain

Summary. Long-chain acyl-CoA hydrolases are a group of enzymes that cleave acyl-CoAs into fatty acids and coenzyme A (CoA-SH). Because acyl-CoAs participate in numerous reactions encompassing lipid synthesis, energy metabolism and regulation, modulating intracellular levels of acyl-CoAs would affect cellular functions. Therefore, acyl-CoA synthetases have been intensively studied. In contrast, acyl-CoA hydrolases have been less investigated, especially in the brain despite the fact that its long-chain acyl-CoA hydrolyzing activity is much higher than that in any other organ in the body. However, recent studies have dissected the multiplicity of this class of enzymes on a genomic basis, and have allowed us to discuss their function. Here, we describe a cytosolic long-chain acyl-CoA hydrolase (referred to as BACH) that is constitutively expressed in the brain, comparing it with other acyl-CoA hydrolases found in peripheral organs that have a role in fatty acid oxidation.     

Localization of a long-chain acyl-CoA hydrolase in spermatogenic cells in mice

Brain acyl-CoA hydrolase (BACH) hydrolyzes long-chain acyl-CoAs to free fatty acids and CoA-SH. BACH is highly distributed in brain and is localized in neurons, but not glial cells. This suggests that BACH plays a specific role in neurons. BACH is also detected in testis, although the expression profile of BACH is unknown in testis. In this study, developmental changes and cellular distribution of BACH were examined in mouse testis. Before postnatal day (P) 10, BACH was detected at very low levels by Western blotting. Then, BACH content rapidly increased from P14 and reached maximum levels at P21, remaining high until at least P70. The increase in BACH content corresponded to the appearance of pachytene spermatocytes, which was confirmed by immunohistochemistry. BACH was also detectable in spermatids, but not in spermatogonia, mature spermatozoa. These results suggest that BACH is expressed in a cell-specific manner and plays a role in spermatogenesis.     

Identification of PTE2, a human peroxisomal long-chain acyl-CoA thioesterase

Computer-based approaches identified PTE2 as a candidate human peroxisomal acyl-CoA thioesterase gene. The PTE2 gene product is highly similar to the rat cytosolic and mitochondrial thioesterases, CTE1 and MTE1, respectively, and terminates in a tripeptide sequence, serine-lysine-valine(COOH), that resembles the consensus sequence for type-1 peroxisomal targeting signals. PTE2 was targeted to peroxisomes and recombinant PTE2 showed intrinsic acyl-CoA thioesterase activity with a pH optimum of 8.5. A comparison of PTE2 and PTE1 thioesterase activities across multiple acyl-CoA substrates indicated that while PTE1 was most active on medium-chain acyl-CoAs, with little activity on long-chain acyl-CoAs, PTE2 displayed high activity on medium- and long-chain acyl-CoAs. The identification of PTE2 therefore offers an explanation for the observed long-chain acyl-CoA thioesterase activity of mammalian peroxisomes.     

Inducible expression of long-chain acyl-CoA hydrolase gene in cell cultures

A long-chain acyl-CoA hydrolase, BACH, is markedly distributed in the brain and localized in neurons. However, the physiological significance of BACH is unclear. To study the gene function, we expressed the mouse BACH gene in C3H 10T1/2 fibroblastic cells using a mifepristone (RU486)-inducible gene expression system. A cell clone, 10T-S6/44, was generated by stable transfection of two plasmids encoding a mifepristone-dependent transactivator and an inducible transgene product, BACH with a C-terminal MYC-tag (BACH-MYC). The transgene expression in the 10T-S6/44 cells was tightly regulated by mifepristone. Induction of BACH-MYC and an increase in palmitoyl-CoA hydrolase activity were observed in the cells treated with 3 × 10^–11 M mifepristone and reached maximal levels at a concentration of 1 × 10^–9 M for 48 h. The growth rate of cells showing the maximal induction of BACH-MYC was reduced, whereas phospholipid synthesis was unchanged. These results suggested that BACH affects specific cellular systems and functions, but not all acyl-CoA-utilizing processes.     

Purification, molecular cloning, and genomic organization of human brain long-chain acyl-CoA hydrolase

An acyl-CoA hydrolase, referred to as hBACH, was purified from human brain cytosol. The enzyme had a molecular mass of 100 kDa and 43-kDa subunits, and was highly active with long-chain acyl-CoAs, e.g. a maximal velocity of 295 micromol/min/mg and K(m) of 6.4 microM for palmitoyl-CoA. Acyl-CoAs with carbon chain lengths of C(8-18) were also good substrates. In human brain cytosol, 85% of palmitoyl-CoA hydrolase activity was titrated by an anti-BACH antibody, which accounted for over 75% of the enzyme activity found in the brain tissue. The cDNA isolated for hBACH, when expressed in Escherichia coli, directed the expression of palmitoyl-CoA hydrolase activity and a 44-kDa protein immunoreactive to the anti-BACH antibody, which in turn neutralized the hydrolase activity. The hBACH cDNA encoded a 338-amino acid sequence which was 95% identical to that of a rat homolog. The hBACH gene spanned about 130 kb and comprised 9 exons, and was mapped to 1p36.2 on the cytogenetic ideogram. These findings indicate that the long-chain acyl-CoA hydrolase present in the brain is well conserved between man and the rat, suggesting a conserved role for this enzyme in the mammalian brain, and enabling genetic studies on the functional analysis of acyl-CoA hydrolase.     

Transcriptional activities of nuclear SREBP-1a, -1c,and -2 to different target promoters of lipogenic and cholesterogenic genes

Recent studies on the in vivo roles of the sterol regulatory element binding protein (SREBP) family indicate that SREBP-2 is more specific to cholesterogenic gene expression whereas SREBP-1 targets lipogenic genes. To define the molecular mechanism involved in this differential regulation, luciferase-reporter gene assays were performed in HepG2 cells to compare the transactivities of nuclear SREBP-1a, -1c, and -2 on a battery of SREBP-target promoters containing sterol regulatory element (SRE), SRE-like, or E-box sequences. The results show first that cholesterogenic genes containing classic SREs in their promoters are strongly and efficiently activated by both SREBP-1a and SREBP-2, but not by SREBP-1c. Second, an E-box containing reporter gene is much less efficiently activated by SREBP-1a and -1c, and SREBP-2 was inactive in spite of its ability to bind to the E-box. Third, promoters of lipogenic enzymes containing variations of SRE (SRE-like sequences) are strongly activated by SREBP-1a, and only modestly and equally by both SREBP-1c and -2. Finally, substitution of the unique tyrosine residue within the basic helix-loop-helix (bHLH) portion of nuclear SREBPs with arginine, the conserved residue found in all other bHLH proteins, abolishes the transactivity of all SREBPs for SRE, and conversely results in markedly increased activity of SREBP-1 but not activity of SREBP-2 for E-boxes. These data demonstrate the different specificity and affinity of nuclear SREBP-1 and -2 for different target DNAs, explaining a part of the mechanism behind the differential in vivo regulation of cholesterogenic and lipogenic enzymes by SREBP-1 and -2, respectively.     

Induction of LPL gene expression by sterols is mediated by a sterol regulatory element and is independent of the presence of multiple E boxes

Overexpression of the adipocyte differentiation and determination factor-1 (ADD-1) or sterol regulatory element binding protein-1 (SREBP-1) induces the expression of numerous genes involved in lipid metabolism, including lipoprotein lipase (LPL). Therefore, we investigated whether LPL gene expression is controlled by changes in cellular cholesterol concentration and determined the molecular pathways involved. Cholesterol depletion of culture medium resulted in a significant induction of LPL mRNA in the 3T3-L1 preadipocyte cell line, whereas addition of cholesterol reduced LPL mRNA expression to basal levels. Similar to the expression of the endogenous LPL gene, the activity of the human LPL gene promoter was enhanced by cholesterol depletion in transient transfection assays, whereas addition of cholesterol caused a reversal of its induction. The effect of cholesterol depletion upon the human LPL gene promoter was mimicked by cotransfection of expression constructs encoding the nuclear form of SREBP-1a, -1c (also called ADD-1) and SREBP-2. Bioinformatic analysis demonstrated the presence of 3 potential sterol regulatory elements (SRE) and 3 ADD-1 binding sequences (ABS), also known as E-box motifs. Using a combination of in vitro protein-DNA binding assays and transient transfection assays of reporter constructs containing mutations in each individual site, a sequence element, termed LPL-SRE2 (SRE2), was shown to be the principal site conferring sterol responsiveness upon the LPL promoter. These data furthermore underscore the importance of SRE sites relative to E-boxes in the regulation of LPL gene expression by sterols and demonstrate that sterols contribute to the control of triglyceride metabolism via binding of SREBP to the LPL regulatory sequences.