The Arabidopsis RING-type E3 ligase XBAT32 mediates the proteasomal degradation of the ethylene biosynthetic enzyme, 1-aminocyclopropane-1-carboxylate synthase 7

E3 ubiquitin ligases select specific proteins for ubiquitin conjugation, and the modified proteins are commonly degraded through the 26S proteasome. XBAT32 is a RING-type E3 ligase involved in maintaining appropriate levels of ethylene. Previous work has suggested that XBAT32 modulates ethylene production by ubiquitinating two ethylene biosynthesis enzymes, ACS4 (type-II isoform) and ACS7 (type-III isoform). In Arabidopsis, conserved sequences within the C-terminal tail of type-I and -II 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) isoforms influence ubiquitin-dependent proteolysis. ACS7, the sole Arabidopsis type-III ACS, contains a truncated C-terminal tail that lacks all known regulatory sequences, which suggests that this isoform may not be subject to ubiquitin-mediated proteasomal degradation. Here we demonstrate in planta that ACS7 is turned over in a 26S proteasome-dependent manner and that degradation of ACS7 requires the E3 ligase XBAT32. Furthermore, the ethylene-related phenotypes that result from overexpression of ACS7 in wild-type plants are greatly exaggerated in xbat32-1, suggesting that XBAT32 is required to attenuate the effect of overexpression of ACS7. This observation is consistent with a role for XBAT32 in the ubiquitin-mediated degradation of ACS7. The dark-grown phenotype of xbat32-1 seedlings overexpressing ACS7 can be effectively rescued by aminoethoxyvinylglycine, an inhibitor of ACS activity. The degradation rate of ACS4 is also significantly slower in the absence of XBAT32, further implicating XBAT32 in the ubiquitin-mediated degradation of ACS4. Altogether, these results demonstrate that XBAT32 targets ethylene biosynthetic enzymes for proteasomal degradation to maintain appropriate levels of hormone production.     

Molecular evolution of the AMP-forming Acetyl-CoA synthetase

Acetyl-CoA-Synthetase (ACS) is involved in the production of acetate, a major metabolite in numerous organisms. There are two forms of this enzyme: ADP-forming ACS and ATP-forming ACS. We focus mainly on the AMP-forming ACS gene, which is relatively well conserved in eubacteria, archeaebacteria, and eukaryotes. BLAST searches in databases showed 30 protein sequences significantly related to the ACS. Most of these sequences were identified as ACS but three of them, belonging to the mammalian species, were annotated as another gene named: the SA gene, which is involved in the essential hypertension. The ACS and SA genes probably derived from a duplication of an ancestral gene but have acquired different functions. Six conserved regions of the ACS protein were defined across the three domains of life. While the precise function of the conserved regions remains unknown, they are probably involved in the enzymatic activity. Among eukaryotes, we found a high variability with respect to the number and the position of introns. However, some positions are conserved between fungi and a nematode. A maximum likelihood tree based upon the conserved regions showed that all sequences except the one from B. subtilis, belong to two basic groups: one the SA-like group including sequences from Archaeoglobus fulgidus and Streptomyces coelicolor, and second, the ACS group. The later can be further divided in two parts: a prokaryotic one including eubacteria and an archaebacterium, and a eukaryotic group within which two proteobacterial sequences branch including ACS from the alpha-proteobacterium Rhodobacter capsulatus. Within the eukaryotic group, bootstrap support is very low, but overall the data are consistent with the view that eukaryotes acquired their ACS gene from the ancestors of mitochondria. The localization of this enzyme in eukaryotic mitochondria is the additional evidence in favor of this interpretation.     

Unique and overlapping expression patterns among the Arabidopsis 1-amino-cyclopropane-1-carboxylate synthase gene family members

1-Aminocyclopropane-1-carboxylate synthase (ACS) catalyzes the rate-limiting step in the ethylene biosynthetic pathway in plants. The Arabidopsis genome encodes nine ACS polypeptides that form eight functional (ACS2, ACS4-9, and ACS11) homodimers and one nonfunctional (ACS1) homodimer. Transgenic Arabidopsis lines were constructed expressing the beta-glucuronidase (GUS) and green fluorescence protein (GFP) reporter genes from the promoter of each of the gene family members to determine their patterns of expression during plant development. All genes, except ACS9, are expressed in 5-d-old etiolated or light-grown seedlings yielding distinct patterns of GUS staining. ACS9 expression is detected later in development. Unique and overlapping expression patterns were detected for all the family members in various organs of adult plants. ACS11 is uniquely expressed in the trichomes of sepals and ACS1 in the replum. Overlapping expression was observed in hypocotyl, roots, various parts of the flower (sepals, pedicle, style, etc.) and in the stigmatic and abscission zones of the silique. Exogenous indole-3-acetic acid (IAA) enhances the constitutive expression of ACS2, 4, 5, 6, 7, 8, and 11 in the root. Wounding of hypocotyl tissue inhibits the constitutive expression of ACS1 and ACS5 and induces the expression of ACS2, 4, 6, 7, 8, and 11. Inducers of ethylene production such as cold, heat, anaerobiosis, and Li(+) ions enhance or suppress the expression of various members of the gene family in the root of light-grown seedlings. Examination of GUS expression in transverse sections of cotyledons reveals that all ACS genes, except ACS9, are expressed in the epidermis cell layer, guard cells, and vascular tissue. Similar analysis with root tip tissue treated with IAA reveals unique and overlapping expression patterns in the various cell types of the lateral root cap, cell division, and cell expansion zones. IAA inducibility is gene-specific and cell type-dependent across the root tip zone. This limited comparative exploration of ACS gene family expression reveals constitutive spatial and temporal expression patterns of all gene family members throughout the growth period examined. The unique and overlapping gene activity pattern detected reveals a combinatorial code of spatio-temporal coexpression among the various gene family members during plant development. This raises the prospect that functional ACS heterodimers may be formed in planta.     

Characterization of metaplastic and heterotopic epithelia in the human gastrointestinal tract by the expression pattern of acyl-CoA synthetase 5

Metaplastic and heterotopic epithelia are frequently found in the human intestine. The recently cloned human acyl-CoA synthetase 5 (ACS5) is a key enzyme in providing cytosolic acyl-CoA thioesters. The aim of the study was to identify and to locate the expression of ACS5 in the gastric body and the small intestine with metaplasia or heterotopia by different methods. In the normal gastrointestinal tract, ACS5 was predominantly found in the villus epithelium of the small intestine, but not in the gastric mucosa. Of note, strong expression of ACS5 was also detectable in intestinal metaplasia of the stomach. Inversely, ACS5 expression could neither be detected in heterotopic gastric mucosa of the corpus type nor in gastric, pseudopyloric, or antral metaplasia of the small intestine. In conclusion, our data implicate that ACS5 is a suitable differentiating marker molecule in the gastrointestinal tract.     

Exon/intron organization and transcription units of the human acyl-CoA synthetase 4 gene

Acyl-CoA synthetase 4 (ACS4) is an arachidonate-preferring isozyme of ACS family predominantly expressed in steroidogenic tissues. Isolation and characterization of genomic clones encoding human ACS4 revealed that the genomic organization of the gene. The human ACS4 gene spans approximately 90 kb and consists of 16 exons. Sequence inspection of the 5'-flanking region revealed potential DNA elements including GATAs, p300, AP-4, SRY, CREB and MyoD. A minimal promoter region required for the expression of ACS4 in HeLa S3 cells was determined. The human ACS4 gene was mapped between the STS markers, WI-17685 and CHLC.GATA81B07 on Xq22-23 region.     

Rat liver acyl-CoA synthetase 4 is a peripheral-membrane protein located in two distinct subcellular organelles,peroxisomes, and mitochondrial-associated membrane

Obesity and non-insulin-dependent diabetes favor storage of fatty acids in triacylglycerol over oxidation. Recently, individual acyl-CoA synthetase (ACS) isoforms have been implicated in the channeling of fatty acids either toward lipid synthesis or toward oxidation. Although ACS1 had been localized to three different subcellular regions in rat liver, endoplasmic reticulum, mitochondria, and peroxisomes, the study had used an antibody raised against the full-length ACS1 protein which cross-reacts with other isoforms, probably because all ACS family members contain highly conserved amino acid sequences. Therefore, we examined the subcellular location of ACS1, ACS4, and ACS5 in rat liver to determine which isoform was present in peroxisomes, whether the ACSs were intrinsic membrane proteins, and which ACS isoforms were up-regulated by PPAR alpha ligands. Non-cross-reacting ACS1, ACS4, and ACS5 peptide antibodies showed that ACS4 was the only ACS isoform present in peroxisomes isolated from livers of gemfibrozil-treated rats. ACS4 was also present in fractions identified as mitochondria-associated membrane (MAM). ACS1 was present in endoplasmic reticulum fractions and ACS5 was present in mitochondrial fractions. Incubation with troglitazone, a specific inhibitor of ACS4, decreased ACS activity in the MAM fractions 30-45% and in the peroxisomal fractions about 30%. Because the signal for ACS4 protein in peroxisomes was so strong compared to the MAM fraction, we examined ACS4 mRNA abundance in livers of rats treated with the PPAR alpha agonist GW9578. Treatment with GW9578 increased ACS4 mRNA abundance 40% and ACS1 mRNA 25%. Although we had originally proposed that ACS4 is linked to triacylglycerol synthesis, it now appears that ACS4 may also be important in activating fatty acids destined for peroxisomal oxidation. We also determined that, unlike ACS1 and 5, ACS4 is not an intrinsic membrane protein. This suggests that ACS4 is probably targeted and linked to MAM and peroxisomes by interactions with other proteins.     

Regulation by adrenocorticotropic hormone and arachidonate of the expression of acyl-CoA synthetase 4, an arachidonate-preferring enzyme expressed in steroidogenic tissues

Acyl-CoA synthetase 4 (ACS4) is an arachidonate-preferring enzyme abundant in steroidogenic tissues. We demonstrate that ACS4 expression in steroidogenic tissues in vivo is induced by adrenocorticotropic hormone (ACTH) and suppressed by glucocorticoid. ACTH also induced ACS4 protein but not its mRNA in Y1 adrenocortical tumor cells, whereas both ACS4 mRNA and protein were increased by dibutyryl cAMP (db-cAMP) and forskolin. Furthermore, the levels of ACS4 mRNA and protein in Y1 cells were induced by arachidonate. These data suggest that ACS4 expression in steroidogenic cells is regulated in coordination with induced steroidogenesis and arachidonate released by cholesterol ester hydrolase.     

Acyl-CoA synthetase isoforms 1, 4, and 5 are present in different subcellular membranes in rat liver and can be inhibited independently

Inhibition studies have suggested that acyl-CoA synthetase (ACS, EC ) isoforms might regulate the use of acyl-CoAs by different metabolic pathways. In order to determine whether the subcellular locations differed for each of the three ACSs present in liver and whether these isoforms were regulated independently, non-cross-reacting peptide antibodies were raised against ACS1, ACS4, and ACS5. ACS1 was identified in endoplasmic reticulum, mitochondria-associated membrane (MAM), and cytosol, but not in mitochondria. ACS4 was present primarily in MAM, and the 76-kDa ACS5 protein was located in mitochondrial membrane. Consistent with these locations, N-ethylmaleimide, an inhibitor of ACS4, inhibited ACS activity 47% in MAM and 28% in endoplasmic reticulum. Troglitazone, a second ACS4 inhibitor, inhibited ACS activity <10% in microsomes and mitochondria and 45% in MAM. Triacsin C, a competitive inhibitor of both ACS1 and ACS4, inhibited ACS activity similarly in endoplasmic reticulum, MAM, and mitochondria, suggesting that a hitherto unidentified triacsin-sensitive ACS is present in mitochondria. ACS1, ACS4, and ACS5 were regulated independently by fasting and re-feeding. Fasting rats for 48 h resulted in a decrease in ACS4 protein, and an increase in ACS5. Re-feeding normal chow or a high sucrose diet for 24 h after a 48-h fast increased both ACS1 and ACS4 protein expression 1.5-2.0-fold, consistent with inhibition studies. These results suggest that ACS1 and ACS4 may be linked to triacylglycerol synthesis. Taken together, the data suggest that acyl-CoAs may be functionally channeled to specific metabolic pathways through different ACS isoforms in unique subcellular locations.     

Functional analyses of two acetyl coenzyme A synthetases in the ascomycete Gibberella zeae

Acetyl coenzyme A (acetyl-CoA) is a crucial metabolite for energy metabolism and biosynthetic pathways and is produced in various cellular compartments with spatial and temporal precision. Our previous study on ATP citrate lyase (ACL) in Gibberella zeae revealed that ACL-dependent acetyl-CoA production is important for histone acetylation, especially in sexual development, but is not involved in lipid synthesis. In this study, we deleted additional acetyl-CoA synthetic genes, the acetyl-CoA synthetases (ACS genes ACS1 and ACS2), to identify alternative acetyl-CoA production mechanisms for ACL. The ACS1 deletion resulted in a defect in sexual development that was mainly due to a reduction in 1-palmitoyl-2-oleoyl-3-linoleoyl-rac-glycerol production, which is required for perithecium development and maturation. Another ACS coding gene, ACS2, has accessorial functions for ACS1 and has compensatory functions for ACL as a nuclear acetyl-CoA producer. This study showed that acetate is readily generated during the entire life cycle of G. zeae and has a pivotal role in fungal metabolism. Because ACSs are components of the pyruvate-acetaldehyde-acetate pathway, this fermentation process might have crucial roles in various physiological processes for filamentous fungi.     

Purification and characterization of two reversible and ADP-dependent acetyl coenzyme A synthetases from the hyperthermophilic archaeon Pyrococcus furiosus.

Pyrococcus furiosus is a strictly anaerobic archaeon (archaebacterium) that grows at temperatures up to 105 degrees C by fermenting carbohydrates and peptides. Cell extracts have been previously shown to contain an unusual acetyl coenzyme A (acetyl-CoA) synthetase (ACS) which catalyzes the formation of acetate and ATP from acetyl-CoA by using ADP and phosphate rather than AMP and PPi. We show here that P. furiosus contains two distinct isoenzymes of ACS, and both have been purified. One, termed ACS I, uses acetyl-CoA and isobutyryl-CoA but not indoleacetyl-CoA or phenylacetyl-CoA as substrates, while the other, ACS II, utilizes all four CoA derivatives. Succinyl-CoA did not serve as a substrate for either enzyme. ACS I and ACS II have similar molecular masses (approximately 140 kDa), and both appear to be heterotetramers (alpha2beta2) of two different subunits of 45 (alpha) and 23 (beta) kDa. They lack metal ions such as Fe2+, Cu2+, Zn2+, and Mg2+ and are stable to oxygen. At 25 degrees C, both enzymes were virtually inactive and exhibited optimal activities above 90 degrees C (at pH 8.0) and at pH 9.0 (at 80 degrees C). The times required to lose 50% of their activity at 80 degrees C were about 18 h for ACS I and 8 h for ACS II. With both enzymes in the acid formation reactions, ADP and phosphate could be replaced by GDP and phosphate but not by CDP and phosphate or by AMP and PPi. The apparent Km values for ADP, GDP, and phosphate were approximately 150, 132, and 396 microM, respectively, for ACS I (using acetyl-CoA) and 61, 236, and 580 microM, respectively, for ACS II (using indoleacetyl-CoA). With ADP and phosphate as substrates, the apparent Km values for acetyl-CoA and isobutyryl-CoA were 25 and 29 microM, respectively, for ACS I and 26 and 12 microM, respectively, for ACS II. With ACS II, the apparent Km value for phenylacetyl-CoA was 4 microM. Both enzymes also catalyzed the reverse reaction, the ATP-dependent formation of the CoA derivatives of acetate (I and II), isobutyrate (I and II), phenylacetate (II only), and indoleacetate (II only). The N-terminal amino acid sequences of the two subunits of ACS I were similar to those of ACS II and to that of a hypothetical 67-kDa protein from Escherichia coli but showed no similarity to mesophilic ACS-type enzymes. To our knowledge, ACS I and II are the first ATP-utilizing enzymes to be purified from a hyperthermophile, and ACS II is the first enzyme of the ACS type to utilize aromatic CoA derivatives.     

The new H2S-releasing compound ACS94 exerts protective effects through the modulation of thiol homoeostasis

The synthesis of a new dithiolethione-cysteine ethyl ester hybrid, ACS94, its metabolites, and its effect on GSH levels in rat tissues and on the concentration of circulating H₂S is described. ACS94 rapidly enters the cells, where it is metabolised to cysteine and the dithiolethione moiety ACS48. Experiments performed through the oral administration of ACS94 to healthy rats showed that it is capable of increasing the GSH levels in most of the analysed organs and the concentration of circulating H₂S. Although the increase in GSH concentration was similar to that obtained by ACS48 and N-acetylcysteine ethyl ester, the H₂S increase was long-lasting and more evident with respect to the parent molecules. Moreover, a decrease of homocysteine in several rat organs and in plasma was noted. This effect may represent a potential therapeutic use of ACS94, as hyperhomocysteinaemia is considered a risk factor for cardiovascular diseases. Lastly, ACS94 was more efficient than N-acetylcysteine in protecting the liver and kidneys against acute acetaminophen toxicity.