ATP-citrate lyase: a mini-review

Obesity is an increasing world problem that may cause several metabolic complications including insulin resistance, hyperlipidemia, hypertension, and atherosclerosis. Development of therapeutic drugs for obesity has been proven difficult. Current strategies for weight reduction are inhibition of food intake through the central nervous system or blocking the absorption of lipids in the gut. These therapies have many side effects, so new treatments are urgently needed. Fat loss could also be achieved through a decrease in the size and number of adipocytes through apoptosis. Apoptosis is a normal phenomenon of cell death for the purpose of maintaining homeostasis. Induction of apoptosis is a reasonable way to remove adipocytes in obese patients. It is reported that several adipokines and natural products play roles in induction of adipocyte apoptosis. Here we review the recent progress of the roles and mechanisms of adipocyte apoptosis induced by leptin, tumor necrosis factor-α (TNF-α), and natural compounds.     

Cloning and expression of a human muscle phosphofructokinase cDNA

The nucleotide sequence of a 2.86-kb cDNA clone containing the complete human muscle phosphofructokinase (PFK) protein-coding region was determined. It comprises 76 bp of 5'-untranslated sequence, 2340 bp encoding human muscle PFK polypeptide, and 399 bp of 3'-untranslated sequence plus a poly(A) tract. A retroviral vector was utilized to express the product of this coding sequence in mouse fibroblasts. The PFK-coding cDNA was shown to code for an enzymatically active polypeptide by immunoprecipitation analysis and DEAE-Sephadex A-25 chromatography.     

Cloning, expression, and cellular localization of a human prenylcysteine lyase

Prenylated proteins contain either a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoid covalently attached to cysteine residues at or near their C terminus. These proteins constitute up to 2% of total cellular protein in eukaryotic cells. The degradation of prenylated proteins raises a metabolic challenge to the cell, because the thioether bond of the modified cysteine is quite stable. We recently identified and isolated an enzyme termed prenylcysteine lyase that cleaves the prenylcysteine to free cysteine and an isoprenoid product (Zhang, L., Tschantz, W. R., and Casey, P. J. (1997) J. Biol. Chem. 272, 23354-23359). To facilitate the molecular characterization of this enzyme, its cloning was undertaken. Overlapping cDNA clones encoding the complete coding sequence of this enzyme were obtained from a human cDNA library. The open reading frame of the gene encoding prenylcysteine lyase is 1515 base pairs and has a nearly ubiquitous expression pattern with a message size of 6 kilobase pairs. Recombinant prenylcysteine lyase was produced in a baculovirus-Sf9 expression system. Analysis of both the recombinant and native enzyme revealed that the enzyme is glycosylated and contains a signal peptide that is cleaved during processing. Additionally, the subcellular localization of this enzyme was determined to be lysosomal. These findings strengthen the notion that prenylcysteine lyase plays an important role in the final step in the degradation of prenylated proteins and will allow further physiological and biochemical characterization of this enzyme.     

Cloning and expression of human apolipoprotein D cDNA

The amino acid sequence of human apolipoprotein D, a component of high density lipoprotein, has been obtained from the cloned cDNA sequence. The 169-amino acid protein has no marked similarity to other apolipoprotein sequences, but has a high degree of homology to plasma retinol-binding protein and other members of the alpha 2u-globulin protein superfamily. Apolipoprotein D mRNA has been detected in human liver, intestine, pancreas, kidney, placenta, adrenal, spleen, and fetal brain tissue. Tissue culture cells transfected with the cloned cDNA secrete material that reacts with anti-apoD antibodies.     

Cloning and expression of the cDNA for human antithrombin III.

A partial cDNA clone for human antithrombin III (ATIII) was obtained by screening a cDNA library prepared from size fractionated liver RNA with a pool of eight 16-base long synthetic DNA fragments whose sequence was determined from protein sequence data. A fragment of the partial cDNA clone was used to enrich RNA for ATIII messages, and cDNA clones encoding the entire ATIII structural gene were identified. The complete nucleotide and predicted amino acid sequences of human ATIII and its 32 residue signal peptide are reported, and provide further opportunity to compare the ATIII primary structure with corresponding regions from homologous proteins, alpha 1-antitrypsin and ovalbumin. Plasmids in which the structural genes for mature and pre-ATIII were linked to the E. coli trp promoter-operator support the synthesis of human antithrombin III and pre-antithrombin III in bacteria.     

Cloning and expression of a novel variant of human interferon-gamma cDNA

A cDNA library was prepared from the poly(A) mRNA isolated from human peripheral blood lymphocytes which were induced by combined treatment with phytohemagglutinin and a phorbol ester. Recombinant plasmids containing human interferon-gamma (HuIFN-gamma) cDNAs were identified by the oligonucleotide-hybridization method. Nucleotide sequence analysis showed that the nucleotide and amino-acid sequences of HuIFN-gamma cDNA in plasmid pIFN gamma-G4 differed from the published data at amino acid position 9 (CAA for glutamine versus AAA for lysine). The cDNA in plasmid pIFN gamma-G4 was expressed under control of the simian virus 40 early promoter in monkey COS cells and a biologically active HuIFN-gamma was secreted from the cells. The cDNA was also inserted into an expression vector carrying an E. coli tryptophan promoter and was expressed in E. coli. The results suggest that the conversion from lysine to glutamine at amino acid position 9 might not affect the specific activity of HuIFN-gamma.     

Cloning and expression of a cDNA encoding a novel human neurotrophic factor

A cDNA encoding a novel human neurotrophic factor (designated nerve growth factor-2; NGF-2) was cloned from a human glioma cDNA library using a synthetic DNA corresponding to human nerve growth factor (NGF). The cloned cDNA encodes a polypeptide composed of 257 amino acid residues including a prepro-sequence of 138 residues and a mature region of 119 residues. The amino acid sequence of human NGF-2 exhibits 58% similarity with that of human NGF. Conditioned medium of COS-7 cells transfected with an expression plasmid for human NGF-2 cDNA supported the survival of sensory neurons isolated from dorsal root ganglia of embryonic chicks. A 1.5 kb of NGF-2 mRNA can be detected from an early development stage in rat brain, by Northern blotting analysis.     

Cloning and expression of the cDNA coding for a human lymphocyte IgE receptor.

Low-affinity receptors (Fc epsilon R) and secreted factors (IgE-BF) which bind to immunoglobulins of the IgE isotype play a key role in the regulation of human IgE synthesis. We report here the cloning of a cDNA coding for the Fc epsilon R of the human B-lymphoblast cell line RPMI 8866. The nucleotide sequence of this cDNA predicts a polypeptide with 321 amino acids and a mol. wt of 36,281 daltons. A functional Fc epsilon R capable of binding IgE was expressed in Chinese hamster ovary cells after stable transformation with the cDNA which had been cloned into a mammalian expression vector. Amino acid sequence analysis of IgE-BF purified from RPMI 8866 cells revealed an amino-terminal sequence of 19 residues which coincides with the predicted amino acid sequence of the Fc epsilon R, starting at residues 148 and 150. A computer search with the translated amino acid sequence of the Fc epsilon R revealed a domain of 120 amino acids having striking homology to the human asialoglycoprotein receptors.     

Cloning of the Trichoderma reesei cDNA encoding a glucuronan lyase belonging to a novel polysaccharide lyase family

The filamentous fungus Trichoderma reesei produces glucuronan lyase (TrGL) when it is grown on beta-(1-->4)-polyglucuronate (cellouronate) as a sole carbon source. The cDNA encoding TrGL was cloned, and the recombinant enzyme was heterologously expressed in Pichia pastoris. The cDNA of TrGL includes a 777-bp open reading frame encoding a 20-amino-acid signal peptide and the 238-amino-acid mature protein. The amino acid sequence showed no similarity to the amino acid sequences of previously described functional proteins, indicating that the enzyme should be classified in a novel polysaccharide lyase (PL) family. Recombinant TrGL catalyzed depolymerization of cellouronate endolytically by beta-elimination and was highly specific for cellouronate. The enzyme was most active at pH 6.5 and 50 degrees C, and its activity and thermostability increased in the presence of Ca2+, suggesting that its calcium dependence is similar to that of other PLs, such as pectate lyases.     

Kinetic and biochemical analyses on the reaction mechanism of a bacterial ATP-citrate lyase.

The prokaryotic ATP-citrate lyase is considered to be a key enzyme of the carbon dioxide-fixing reductive tricarboxylic acid (RTCA) cycle. Kinetic examination of the ATP-citrate lyase from the green sulfur bacterium Chlorobium limicola (Cl-ACL), an alpha(4)beta(4) heteromeric enzyme, revealed that the enzyme displayed typical Michaelis-Menten kinetics toward ATP with an apparent K(m) value of 0.21 +/- 0.04 mm. However, strong negative cooperativity was observed with respect to citrate binding, with a Hill coefficient (n(H)) of 0.45. Although the dissociation constant of the first citrate molecule was 0.057 +/- 0.008 mm, binding of the first citrate molecule to the enzyme drastically decreased the affinity of the enzyme for the second molecule by a factor of 23. ADP was a competitive inhibitor of ATP with a K(i) value of 0.037 +/- 0.006 mm. Together with previous findings that the enzyme catalyzed the reaction only in the direction of citrate cleavage, these kinetic features indicated that Cl-ACL can regulate both the direction and carbon flux of the RTCA cycle in C. limicola. Furthermore, in order to gain insight on the reaction mechanism, we performed biochemical analyses of Cl-ACL. His273 of the alpha subunit was indicated to be the phosphorylated residue in the catalytic center, as both catalytic activity and phosphorylation of the enzyme by ATP were abolished in an H273A mutant enzyme. We found that phosphorylation of the subunit was reversible. Nucleotide preference for activity was in good accordance with the preference for phosphorylation of the enzyme. Although residues interacting with nucleotides in the succinyl-CoA synthetase from Escherichia coli were conserved in AclB, AclA alone could be phoshorylated with the same nucleotide specificity observed in the holoenzyme. However, AclB was necessary for enzyme activity and contributed to enhance phosphorylation and stabilization of AclA.     

弗氏柠檬酸细菌酪氨酸酚解酶基因在大肠杆菌中的克隆与表达

以基因组DNA为模板,利用PCR技术从弗氏柠檬酸细菌（Citrobacter freundii)中扩增得到含有酪氨酸酚解酶基因的DNA片段,定向连续到质粒pUC118上,得到重组质粒pTPL,将此重组质粒转化到受体菌E.colXL-1-Blue MRF′中,通过蓝白斑鉴定挑出阳性菌株。从此阳性菌株中提取质粒pTPL并将此质粒转入到E.coliJM109中,用E.coliJM109（pTPL)制备高活性的酪氨酸酚解酶。对质粒稳定性的研究表明,E.coliJM109（pTPL)在无选择压力下37℃连续培养50代以上,质粒丢失率仅有15％,说明质粒基本稳定。     

ATP-citrate lyase from rat liver. Characterisation of the citryl-enzyme complexes.

The mechanism of ATP-citrate lyase has been proposed to involve a citryl-enzyme intermediate. When the enzyme is incubated with its substrates ATP and [14C]citrate, but in the absence of the final acceptor, two distinct types of citrate-containing complex can be isolated. At early time points, a highly unstable complex can be isolated by gel filtration which has a half-life of 36 s at 25 degrees C. This complex reacts rapidly with CoA, but cannot be acid-precipitated; behaviour consistent with its identification as enzyme-citryl phosphate. However, ATP-citrate lyase is also capable of undergoing a slow time-dependent covalent incorporation of radiolabel from [14C]citrate. This modification is acid-stable, non-specific, and cannot be reversed by the addition of CoA. When cytochrome is included in the reaction mixture as a heterologous acceptor, it is also citrylated. These reactions require that when ATP-citrate lyase is incubated with all its substrates except for CoA, a freely diffusible citrylating species is generated within the active site. This evidence suggests that there is no requirement for the mechanism of ATP-citrate lyase to proceed via a covalent citryl-enzyme intermediate. By analogy with succinyl-CoA synthetase, an enzyme which has a high degree of sequence similarity with ATP-citrate lyase, a simple mechanism is proposed for the enzyme in which citryl-CoA is produced by direct nucleophilic attack on citryl phosphate.     

Cloning and expression of isocitrate lyase from human round worm Strongyloides stercoralis

A full length cDNA (1463 bp) encoding isocitrate lyase (EC 4.1.3.1) of Strongyloides stercoralis is described. The nucleotide sequence of this insert identified a cDNA coding for the isocitrate lyase. The conceptually translated amino acid sequence of the open reading frame for S. stercoralis isocitrate lyase encodes a 450 amino acid residue protein with an apparent molecular weight of 50 kDa and a predicted pl of 6.39. The sequence is 69% A/T, reflecting a characteristic A/T codon bias of S. stercoralis. The amino acid sequence of S. stercoralis isocitrate lyase is compared with bifunctional glyoxylate cycle protein of Caenorhabditis elegans and isocitrate lyases from Chlamydomonas reinhardtii and Myxococcus xanthus. The full length cDNA of S. stercoralis was expressed in pRSET vector and bacteriophage T7 promoter based expression system. S. stercoralis lyase recombinant protein, purified via immobilized metal affinity chromatography, showed a molecular mass of 50 kDa on polyacrylamide gels. The role of isocitrate lyase in the glyoxylate cycle and energy metabolism of S. stercoralis is also discussed.     

Physiological characterization of ATP-citrate lyase in Aspergillus niger

Acetyl-CoA, an important molecule in cellular metabolism, is generated in multiple subcellular compartments and mainly used for energy production, biosynthesis of a diverse set of molecules, and protein acetylation. In eukaryotes, cytosolic acetyl-CoA is derived mainly from the conversion of citrate and CoA by ATP-citrate lyase. Here, we describe the targeted deletions of acl1 and acl2, two tandem divergently transcribed genes encoding subunits of ATP-citrate lyase in Aspergillus niger. We show that loss of acl1 or/and acl2 results in a significant decrease of acetyl-CoA and citric acid levels in these mutants, concomitant with diminished vegetative growth, decreased pigmentation, reduced asexual conidiogenesis, and delayed conidial germination. Exogenous addition of acetate repaired the defects of acl-deficient strains in growth and conidial germination but not pigmentation and conidiogenesis. We demonstrate that both Acl1 and Acl2 subunits are required to form a functional ATP-citrate lyase in A. niger. First, deletion of acl1 or/and acl2 resulted in similar defects in growth and development. Second, enzyme activity assays revealed that loss of either acl1 or acl2 gene resulted in loss of ATP-citrate lyase activity. Third, in vitro enzyme assays using bacterially expressed 6His-tagged Acl protein revealed that only the complex of Acl1 and Acl2 showed ATP-citrate lyase activity, no enzyme activities were detected with the individual protein. Fourth, EGFP-Acl1 and mCherry-Acl2 proteins were co-localized in the cytosol. Thus, acl1 and acl2 coordinately modulate the cytoplasmic acetyl-CoA levels to regulate growth, development, and citric acid synthesis in A. niger.     

ATP-citrate lyase as a substrate of protein histidine phosphatase in vertebrates

The first protein histidine phosphatase from vertebrates discovered recently was found in a variety of tissues, however, a physiological substrate protein was missing. Phosphorylation of liver extracts in the presence of EDTA, followed by SDS-PAGE and autoradiography showed labeling of three proteins. Acid- and alkaline-treatment revealed the existence of N-phosphates. Addition of histidine phosphatase exclusively resulted in dephosphorylation of a 110kDa protein (denaturing conditions). Gelfiltration revealed its native molecular mass of approximately 450kDa. That protein was purified and identified as ATP-citrate lyase. The results are in favor of histidine phosphatase playing an important yet unidentified role in metabolic processes.     

Cloning of a human liver microsomal UDP-glucuronosyltransferase cDNA.

A cDNA clone (HLUG 25) encoding the complete sequence of a human liver UDP-glucuronosyltransferase was isolated from a lambda gt11 human liver cDNA library. The library was screened by hybridization to a partial-length human UDP-glucuronosyltransferase cDNA (pHUDPGT1) identified from a human liver pEX cDNA expression library by using anti-UDP-glucuronosyltransferase antibodies. The authenticity of the cDNA clone was confirmed by hybrid-select translation and extensive sequence homology to rat liver UDP-glucuronosyltransferase cDNAs. The sequence of HLUG 25 cDNA was determined to be 2104 base-pairs long, including a poly(A) tail, and contains a long open reading frame. The possible site of translation initiation of this sequence is discussed with reference to a rat UDP-glucuronosyltransferase cDNA clone (RLUG 38).     

Cloning and expression of human cDNA encoding human homologue of pituitary tumor transforming gene.

Recently, a potent transforming gene which was exclusively expressed in rat pituitary tumor but not in normal pituitary had been isolated and named as pituitary tumor transforming gene (PTTG). A cDNA clone encoding human homologue of rat PTTG was isolated from human fetal liver cDNA library. It contained an open reading frame of 603 base pairs predicting a protein composed of 201 amino acids with a calculated molecular weight of 26 kDa. The deduced protein showed about 85% homology (78% identity, 7% favored substitution) with the rat PTTG. Northern blot analysis showed that the cDNA hybridized to 1.0 kb mRNA species which was expressed in fetal liver and several cancer cell lines. These results suggest that the presence of the human homologue of rat PTTG gene may not be restricted to pituitary tumor.     

Cloning and functional expression of human cDNA for the ETB endothelin receptor.

We report the cloning of human cDNA encoding an ETB (non-isopeptide-selective) subtype of the endothelin receptor. The predicted amino acid sequence of the human ETB endothelin receptor was 87.8% and 62.9% identical with the previously cloned rat ETB and ETA receptors, respectively. COS cells transiently transfected with the cloned cDNA expressed specific, high-affinity binding sites for endothelin isopeptides and responded to the peptides with a transient increase of [Ca2+]i; endothelin-1 and endothelin-3 exhibited approximately equal potencies both in displacing 125I-labeled endothelin-1 binding and in eliciting [Ca2+]i transients. The ETB receptor mRNAs were expressed in various human tissues and also in the intact porcine aortic intimal cells ex vivo.