ACC2 is under-expressed in lung adenocarcinoma and predicts poor clinical outcomes.

PURPOSE: Acetyl-CoA Carboxylases (ACCs) are key fatty acid metabolic enzymes responsible for catalyzing the carboxylation of acetyl-CoA to malonyl-CoA. The role of ACC1 has been associated with tumor biology, but the role of ACC2 in cancer remains largely uncharacterized. METHODS: We conducted a transcriptomic analysis using GEPIA and Oncomine to study the expression of ACC2 in different cancers. Immunohistochemistry was used to examine the expression of ACC2 in lung cancer tissue microarray, and the correlation between ACC2 expression and clinical parameters was analyzed. Following ACC2 knockdown by RNA interference in A549 and HCC827 cells, Cell Counting Kit­8 and transwell assays were used to detect cell proliferation and migration. Real-time PCR was used to detect cell cycle-related genes in A549 cells. GEO dataset and KM-plotter database were used to analyze the relationship between ACC2 expression and the prognosis in lung cancer patients. RESULTS: We found that ACC2 is under-expressed in cancerous tissue and the expression of ACC2 is negatively correlated with tumor size, regional lymph-node metastases, and clinical stage of lung adenocarcinoma patients. In addition, knocking down ACC2 in A549 cells and HCC827 cells can promote cell proliferation and migration, and cell cycle-related genes MAD2L1 and CCNB2 were up-regulated after ACC2 was knockdown in A549 cells. Finally, we found that lung adenocarcinoma patients with under-expressed ACC2 have a worse prognosis. CONCLUSIONS: Our results suggest that ACC2 is a potential diagnostic and prognostic marker that negatively correlated with clinical outcomes in lung adenocarcinoma.

Recent development in acetyl-CoA carboxylase inhibitors and their potential as novel drugs.

Acetyl-CoA carboxylase (ACC), a critical enzyme in the regulation of fatty acid synthesis and metabolism, has emerged as an attractive target for a plethora of emerging diseases, such as diabetes mellitus, nonalcoholic fatty liver disease, cancer, bacterial infections and so on. With decades of efforts in medicinal chemistry, significant progress has been made toward the design and discovery of a considerable number of inhibitors of this enzyme. In this review, we not only clarify the role of ACC in emerging diseases, but also summarize recent developments of potent ACC inhibitors and discuss their molecular mechanisms of action and potentials as novel drugs as well as future perspectives toward the design and discovery of novel ACC inhibitors.

Computational and experimental investigations on the interactions of aryloxy-phenoxy-propionate herbicides to estrogen receptor alpha in zebrafish.

When the amount of pesticide exceeds the self-purification ability of the environment, it will be enriched in the human body through the atmosphere, soil, water circulation, etc., threatening human health. Aryloxy-phenoxy-propionate (APP) herbicides are a class of acetyl-CoA carboxylase (ACCase) inhibitor herbicides, widely used in field-weeding of soybean, cabbage, peanut and other crops. However, due to the water circulation, surface runoff and the agronomic practices such as watering irrigation, APP herbicides have the risk of polluting water and destroying the living environment of aquatic organisms. In this paper, a multistep framework combining homology modeling, molecular docking and molecular dynamic simulations were adopted to explore the interactions between APP herbicides and zebrafish estrogen receptor α (ERα) to investigate the estrogenic activities of the herbicides. The structure of zebrafish ERα was modeled by homology modeling, using the human's estrogen receptor α (PDB ID:2YJA) as the template. Then, eight typical APP herbicides were selected to dock with the zebrafish ERα, and it was determined that there were clear interactions between the herbicides and the receptor. The binding patterns of Quizalofop-P-ethyl (QPE), Clodinafop-propargyl (CP) and Haloxyfop-P (HP) with ERα were further investigated by molecular dynamics and binding free energy calculation. The results showed the van der Waals force and electrostatic force were the main driving forces for maintaining the stability of the complex system. In order to verify the theoretical prediction, an exposed experiment was conducted to study the effects of different concentrations of herbicides on VTG level of zebrafish in vivo and the results were consistent with the computational method. The results of this study revealed the mechanism of the action between APP herbicides and zebrafish estrogen receptors, and also provided ideas for optimizing the herbicides.

Human Acetyl-CoA Carboxylase 1 Is an Isomerase: Carboxyl Transfer Is Activated by Catalytic Effect of Isomerization.

Obesity and its related diseases such as cancer and diabetes are leading life-threatening issues in the modern world. Thus, new drugs toward obesity and obesity-caused diseases are highly desired. Human acetyl-CoA carboxylase 1 (hACC1) in charge of the rate-limiting step of the human fatty acid synthesis was recognized as an attractive target for rational drug design. The fundamental reaction mechanism and nature of the transition state of hACC1 remain unclear. In this study, combined quantum mechanics and molecular mechanics (QM/MM), molecular dynamics (MD), and free-energy simulations were performed to investigate the catalytic mechanism of the hACC1-catalyzed carboxyl-transfer reaction. Our computational results show a three-step mechanism for carboxyl transferase (CT)-catalyzed reaction, including isomerization of carboxybiotin, proton-transfer from acetyl-CoA to carboxybiotin, and carboxylation of acetyl-CoA enolate. Interestingly, isomerization of carboxybiotin is the rate-limiting step of the entire reaction pathway, indicating hACC1 has the catalytic effect of isomerization and thus might be an isomerase also. The activation free-energy barrier of carboxyl-transfer catalyzed by hACC1 was calculated to be 16.4 kcal/mol, in excellent agreement with the experimental result (16.7 kcal/mol). The obtained reaction mechanism together with the nature of the transition state provides helpful knowledge not only for future investigation of other ACCs but also for rational design of hACC1 inhibitors, such as TS analogue. The catalytic effect of hACC1 isomerization is discussed.

Structural basis for regulation of human acetyl-CoA carboxylase.

Acetyl-CoA carboxylase catalyses the ATP-dependent carboxylation of acetyl-CoA, a rate-limiting step in fatty acid biosynthesis1,2. Eukaryotic acetyl-CoA carboxylases are large, homodimeric multienzymes. Human acetyl-CoA carboxylase occurs in two isoforms: the metabolic, cytosolic ACC1, and ACC2, which is anchored to the outer mitochondrial membrane and controls fatty acid ß-oxidation1,3. ACC1 is regulated by a complex interplay of phosphorylation, binding of allosteric regulators and protein-protein interactions, which is further linked to filament formation1,4-8. These filaments were discovered in vitro and in vivo 50 years ago7,9,10, but the structural basis of ACC1 polymerization and regulation remains unknown. Here, we identify distinct activated and inhibited ACC1 filament forms. We obtained cryo-electron microscopy structures of an activated filament that is allosterically induced by citrate (ACC-citrate), and an inactivated filament form that results from binding of the BRCT domains of the breast cancer type 1 susceptibility protein (BRCA1). While non-polymeric ACC1 is highly dynamic, filament formation locks ACC1 into different catalytically competent or incompetent conformational states. This unique mechanism of enzyme regulation via large-scale conformational changes observed in ACC1 has potential uses in engineering of switchable biosynthetic systems. Dissecting the regulation of acetyl-CoA carboxylase opens new paths towards counteracting upregulation of fatty acid biosynthesis in disease.

Calcineurin-mediated Dephosphorylation of Acetyl-coA Carboxylase is Required for Pheromone Biosynthesis Activating Neuropeptide (PBAN)-induced Sex Pheromone Biosynthesis in Helicoverpa armigera.

Chemical signaling plays a critical role in the behavior and physiology of many animals. Female insects, as many other animals, release sex pheromones to attract males for mating. The evolutionary and ecological success of insects therefore hinges on their ability to precisely mediate (including initiation and termination) pheromone biosynthesis. Pheromone biosynthesis activating neuropeptide (PBAN) acts directly on pheromone glands to regulate sex pheromone production using Ca2+ and cyclic-AMP as secondary messengers in the majority of species. However, the molecular mechanism downstream of the secondary messengers has not yet been elucidated in heliothine species. The present study shows that calcineurin, protein kinase A (PKA) and acetyl-coA carboxylase (ACC) are key components involved in PBAN-induced sex pheromone biosynthesis in Helicoverpa armigera using PBAN-dependent phosphoproteomics in combination with transcriptomics. RNAi-mediated knockdown and inhibitor assay demonstrated that calcineurin A is required for PBAN-induced ACC activation and sex pheromone production. Calcineurin-dependent phosphoproteomics and in vitro calcineurin phosphorylation assay further revealed that calcineurin regulated ACC activity by dephosphorylating ser84 and ser92. In addition, PKA-dependent phosphoproteomics and activity analysis revealed that PKA reduces the activity of AMP-activated protein kinase (AMPK), a negative regulator of ACC by phosphorylating the conserved ser92. Taken together, our findings indicate that calcineurin acts as the downstream signal of PBAN/G-protein receptor/Ca2+ to activate ACC through dephosphorylation while inactivating AMPK via PKA to reduce ACC phosphorylation, thus facilitating calcineurin activation of ACC.

AMP-activated protein kinase (AMPK) activator A-769662 increases intracellular calcium and ATP release from astrocytes in an AMPK-independent manner.

AIM: To test the hypothesis that, given the role of AMP-activated protein kinase (AMPK) in regulating intracellular ATP levels, AMPK may alter ATP release from astrocytes, the main sources of extracellular ATP (eATP) within the brain. MATERIALS AND METHODS: Measurements of ATP release were made from human U373 astrocytoma cells, primary mouse hypothalamic (HTAS) and cortical astrocytes (CRTAS) and wild-type and AMPK α1/α2 null mouse embryonic fibroblasts (MEFs). Cells were treated with drugs known to modulate AMPK activity: A-769662, AICAR and metformin, for up to 3 hours. Intracellular calcium was measured using Fluo4 and Fura-2 calcium-sensitive fluorescent dyes. RESULTS: In U373 cells, A-769662 (100 µM) increased AMPK phosphorylation, whereas AICAR and metformin (1 mM) induced a modest increase or had no effect, respectively. Only A-769662 increased eATP levels, and this was partially blocked by AMPK inhibitor Compound C. A-769662-induced increases in eATP were preserved in AMPK α1/α2 null MEF cells. A-769662 increased intracellular calcium in U373, HTAS and CRTAS cells and chelation of intracellular calcium using BAPTA-AM reduced A-769662-induced eATP levels. A-769662 also increased ATP release from a number of other central and peripheral endocrine cell types. CONCLUSIONS: AMPK is required to maintain basal eATP levels but is not required for A-769662-induced increases in eATP. A-769662 (>50 µM) enhanced intracellular calcium levels leading to ATP release in an AMPK and purinergic receptor independent pathway.

PHD3 Loss in Cancer Enables Metabolic Reliance on Fatty Acid Oxidation via Deactivation of ACC2.

While much research has examined the use of glucose and glutamine by tumor cells, many cancers instead prefer to metabolize fats. Despite the pervasiveness of this phenotype, knowledge of pathways that drive fatty acid oxidation (FAO) in cancer is limited. Prolyl hydroxylase domain proteins hydroxylate substrate proline residues and have been linked to fuel switching. Here, we reveal that PHD3 rapidly triggers repression of FAO in response to nutrient abundance via hydroxylation of acetyl-coA carboxylase 2 (ACC2). We find that PHD3 expression is strongly decreased in subsets of cancer including acute myeloid leukemia (AML) and is linked to a reliance on fat catabolism regardless of external nutrient cues. Overexpressing PHD3 limits FAO via regulation of ACC2 and consequently impedes leukemia cell proliferation. Thus, loss of PHD3 enables greater utilization of fatty acids but may also serve as a metabolic and therapeutic liability by indicating cancer cell susceptibility to FAO inhibition.

Hybrid Structure of a Dynamic Single-Chain Carboxylase from Deinococcus radiodurans.

Biotin-dependent acyl-coenzyme A (CoA) carboxylases (aCCs) are involved in key steps of anabolic pathways and comprise three distinct functional units: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyl transferase (CT). YCC multienzymes are a poorly characterized family of prokaryotic aCCs of unidentified substrate specificity, which integrate all functional units into a single polypeptide chain. We employed a hybrid approach to study the dynamic structure of Deinococcus radiodurans (Dra) YCC: crystal structures of isolated domains reveal a hexameric CT core with extended substrate binding pocket and a dimeric BC domain. Negative-stain electron microscopy provides an approximation of the variable positioning of the BC dimers relative to the CT core. Small-angle X-ray scattering yields quantitative information on the ensemble of Dra YCC structures in solution. Comparison with other carrier protein-dependent multienzymes highlights a characteristic range of large-scale interdomain flexibility in this important class of biosynthetic enzymes.

The dynamic organization of fungal acetyl-CoA carboxylase.

Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control.

Crystal structure of the 500-kDa yeast acetyl-CoA carboxylase holoenzyme dimer.

Acetyl-CoA carboxylase (ACC) has crucial roles in fatty acid metabolism and is an attractive target for drug discovery against diabetes, cancer and other diseases. Saccharomyces cerevisiae ACC (ScACC) is crucial for the production of very-long-chain fatty acids and the maintenance of the nuclear envelope. ACC contains biotin carboxylase (BC) and carboxyltransferase (CT) activities, and its biotin is linked covalently to the biotin carboxyl carrier protein (BCCP). Most eukaryotic ACCs are 250-kilodalton (kDa), multi-domain enzymes and function as homodimers and higher oligomers. They contain a unique, 80-kDa central region that shares no homology with other proteins. Although the structures of the BC, CT and BCCP domains and other biotin-dependent carboxylase holoenzymes are known, there is currently no structural information on the ACC holoenzyme. Here we report the crystal structure of the full-length, 500-kDa holoenzyme dimer of ScACC. The structure is remarkably different from that of the other biotin-dependent carboxylases. The central region contains five domains and is important for positioning the BC and CT domains for catalysis. The structure unexpectedly reveals a dimer of the BC domain and extensive conformational differences compared to the structure of the BC domain alone, which is a monomer. These structural changes reveal why the BC domain alone is catalytically inactive and define the molecular mechanism for the inhibition of eukaryotic ACC by the natural product soraphen A and by phosphorylation of a Ser residue just before the BC domain core in mammalian ACC. The BC and CT active sites are separated by 80 Å, and the entire BCCP domain must translocate during catalysis.

Acetyl CoA carboxylase inactivation and meiotic maturation in mouse oocytes.

In mouse oocytes, meiotic induction by pharmacological activation of PRKA (adenosine monophosphate-activated protein kinase; formerly known as AMPK) or by hormones depends on stimulation of fatty acid oxidation (FAO). PRKA stimulates FAO by phosphorylating and inactivating acetyl CoA carboxylase (ACAC; formerly ACC), leading to decreased malonyl CoA levels and augmenting fatty-acid transport into mitochondria. We investigated a role for ACAC inactivation in meiotic resumption by testing the effect of two ACAC inhibitors, CP-640186 and Soraphen A, on mouse oocytes maintained in meiotic arrest in vitro. These inhibitors significantly stimulated the resumption of meiosis in arrested cumulus cell-enclosed oocytes, denuded oocytes, and follicle-enclosed oocytes. This stimulation was accompanied by an increase in FAO. Etomoxir, a malonyl CoA analogue, prevented meiotic resumption as well as the increase in FAO induced by ACAC inhibition. Citrate, an ACAC activator, and CBM-301106, an inhibitor of malonyl CoA decarboxylase, which converts malonyl CoA to acetyl CoA, suppressed both meiotic induction and FAO induced by follicle-stimulating hormone, presumably by maintaining elevated malonyl CoA levels. Mouse oocyte-cumulus cell complexes contain both isoforms of ACAC (ACACA and ACACB); when wild-type and Acacb(-/-) oocytes characteristics were compared, we found that these single-knockout oocytes showed a significantly higher FAO level and a reduced ability to maintain meiotic arrest, resulting in higher rates of germinal vesicle breakdown. Collectively, these data support the model that ACAC inactivation contributes to the maturation-promoting activity of PRKA through stimulation of FAO.

PT-1 selectively activates AMPK-γ1 complexes in mouse skeletal muscle, but activates all three γ subunit complexes in cultured human cells by inhibiting the respiratory chain.

AMP-activated protein kinase (AMPK) occurs as heterotrimeric complexes in which a catalytic subunit (α1/α2) is bound to one of two ß subunits (ß1/ß2) and one of three γ subunits (γ1/γ2/γ3). The ability to selectively activate specific isoforms would be a useful research tool and a promising strategy to combat diseases such as cancer and Type 2 diabetes. We report that the AMPK activator PT-1 selectively increased the activity of γ1- but not γ3-containing complexes in incubated mouse muscle. PT-1 increased the AMPK-dependent phosphorylation of the autophagy-regulating kinase ULK1 (unc-51-like autophagy-activating kinase 1) on Ser555, but not proposed AMPK-γ3 substrates such as Ser231 on TBC1 (tre-2/USP6, BUB2, cdc16) domain family, member 1 (TBC1D1) or Ser212 on acetyl-CoA carboxylase subunit 2 (ACC2), nor did it stimulate glucose transport. Surprisingly, however, in human embryonic kidney (HEK) 293 cells expressing human γ1, γ2 or γ3, PT-1 activated all three complexes equally. We were unable to reproduce previous findings suggesting that PT-1 activates AMPK by direct binding between the kinase and auto-inhibitory domains (AIDs) of the α subunit. We show instead that PT-1 activates AMPK indirectly by inhibiting the respiratory chain and increasing cellular AMP:ATP and/or ADP:ATP ratios. Consistent with this mechanism, PT-1 failed to activate AMPK in HEK293 cells expressing an AMP-insensitive R299G mutant of AMPK-γ1. We propose that the failure of PT-1 to activate γ3-containing complexes in muscle is not an intrinsic feature of such complexes, but is because PT-1 does not increase cellular AMP:ATP ratios in the specific subcellular compartment(s) in which γ3 complexes are located.

A dihydrochalcone and several homoisoflavonoids from Polygonatum odoratum are activators of adenosine monophosphate-activated protein kinase.

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a major cellular energy sensor and master regulator of metabolic homeostasis; thus, AMPK plays a central role in studies on diabetes and related metabolic diseases. From the rhizomes of Polygonatum odoratum (Mill.) Druce, six homoisoflavonoids (1-6) and one dihydrochalcone (7) were isolated, and the structures of polygonatones A-D (4-7) were elucidated by various spectroscopic analyses. Compounds 1-7 were evaluated for their effect on AMPK activation. The amount of active phosphorylated AMPK and acetyl-CoA carboxylase in rat liver epithelial IAR-20 cells increased when the cells were incubated with the aforementioned compounds. Specifically, (3R)-5,7-dihydroxyl-6-methyl-8-methoxyl-3-(4'-hydroxylbenzyl)-chroman-4-one (1), (3R)-5,7-dihydroxyl-6,8-dimethyl-3-(4'-hydroxylbenzyl)-chroman-4-one (2), (3R)-5,7-dihydroxyl-6-methyl-3-(4'-hydroxylbenzyl)-chroman-4-one (3), and polygonatone D (7) exhibited significant activation effects.

Potential functional replacement of the plastidic acetyl-CoA carboxylase subunit (accD) gene by recent transfers to the nucleus in some angiosperm lineages.

Eukaryotic cells originated when an ancestor of the nucleated cell engulfed bacterial endosymbionts that gradually evolved into the mitochondrion and the chloroplast. Soon after these endosymbiotic events, thousands of ancestral prokaryotic genes were functionally transferred from the endosymbionts to the nucleus. This process of functional gene relocation, now rare in eukaryotes, continues in angiosperms. In this article, we show that the chloroplastic acetyl-CoA carboxylase subunit (accD) gene that is present in the plastome of most angiosperms has been functionally relocated to the nucleus in the Campanulaceae. Surprisingly, the nucleus-encoded accD transcript is considerably smaller than the plastidic version, consisting of little more than the carboxylase domain of the plastidic accD gene fused to a coding region encoding a plastid targeting peptide. We verified experimentally the presence of a chloroplastic transit peptide by showing that the product of the nuclear accD fused to green fluorescent protein was imported in the chloroplasts. The nuclear gene regulatory elements that enabled the erstwhile plastidic gene to become functional in the nuclear genome were identified, and the evolution of the intronic and exonic sequences in the nucleus is described. Relocation and truncation of the accD gene is a remarkable example of the processes underpinning endosymbiotic evolution.

Chemical genetics of acetyl-CoA carboxylases.

Chemical genetic studies on acetyl-CoA carboxylases (ACCs), rate-limiting enzymes in long chain fatty acid biosynthesis, have greatly advanced the understanding of their biochemistry and molecular biology and promoted the use of ACCs as targets for herbicides in agriculture and for development of drugs for diabetes, obesity and cancers. In mammals, ACCs have both biotin carboxylase (BC) and carboxyltransferase (CT) activity, catalyzing carboxylation of acetyl-CoA to malonyl-CoA. Several classes of small chemicals modulate ACC activity, including cellular metabolites, natural compounds, and chemically synthesized products. This article reviews chemical genetic studies of ACCs and the use of ACCs for targeted therapy of cancers.

Structure and function of biotin-dependent carboxylases.

Biotin-dependent carboxylases include acetyl-CoA carboxylase (ACC), propionyl-CoA carboxylase (PCC), 3-methylcrotonyl-CoA carboxylase (MCC), geranyl-CoA carboxylase, pyruvate carboxylase (PC), and urea carboxylase (UC). They contain biotin carboxylase (BC), carboxyltransferase (CT), and biotin-carboxyl carrier protein components. These enzymes are widely distributed in nature and have important functions in fatty acid metabolism, amino acid metabolism, carbohydrate metabolism, polyketide biosynthesis, urea utilization, and other cellular processes. ACCs are also attractive targets for drug discovery against type 2 diabetes, obesity, cancer, microbial infections, and other diseases, and the plastid ACC of grasses is the target of action of three classes of commercial herbicides. Deficiencies in the activities of PCC, MCC, or PC are linked to serious diseases in humans. Our understanding of these enzymes has been greatly enhanced over the past few years by the crystal structures of the holoenzymes of PCC, MCC, PC, and UC. The structures reveal unanticipated features in the architectures of the holoenzymes, including the presence of previously unrecognized domains, and provide a molecular basis for understanding their catalytic mechanism as well as the large collection of disease-causing mutations in PCC, MCC, and PC. This review will summarize the recent advances in our knowledge on the structure and function of these important metabolic enzymes.

Targeting acetyl-CoA carboxylases: small molecular inhibitors and their therapeutic potential.

Acetyl-CoA carboxylases (ACCs) play a rate-limiting role in fatty acid biosynthesis in plants, microbes, mammals and humans. ACCs have the activity of both biotin carboxylase (BC) and carboxyltransferase (CT), catalyzing carboxylation of Acetyl-CoA to malonyl-CoA. In the past years, ACCs have been used as targets for herbicides in agriculture and for drug discovery and development of human diseases, such as microbial infections, diabetes, obesity and cancer. A great number of small molecule ACC inhibitors have been developed, including natural and non-natural (artificial) products. These chemicals target BC reaction, CT reaction or ACC phosphorylation. This article provides a comprehensive review and updates of ACC inhibitors, with a focus on their therapeutic application in metabolic syndromes and malignant diseases. The patent status of common ACC inhibitors is discussed.

Phosphoproteomic analysis of mouse thymoma cells treated with tributyltin oxide: TBTO affects proliferation and energy sensing pathways.

We report the results of phosphoproteomic analysis of mouse thymoma cells treated with tributyltin oxide (TBTO), an immunotoxic compound. After cell lysis, phosphoproteins were isolated using Phosphoprotein Purification Kit, separated by SDS-PAGE and subsequently digested with trypsin. Phosphopeptides were enriched employing titanium dioxide, and the obtained fractions were analyzed by nano-LC-MS/MS. A total of 160 phosphoproteins and 328 phosphorylation sites were identified in thymoma cells. Among the differentially phosphorylated proteins identified in TBTO-treated cells were key enzymes, which catalyze rate-limiting steps in pathways that are sensitive to cellular energy status. These proteins included acetyl-CoA carboxylase isoform 1, which catalyzes the rate-limiting step of fatty acid synthesis. Another enzyme was glutamine: fructose-6-phosphate amidotransferase, GFAT1, the first and rate-limiting enzyme for the hexoamine synthesis pathway. Pyruvate dehydrogenase (PDH), a multicomplex enzyme that catalyzes the rate-limiting step of aerobic oxidation of fuel carbohydrates, was identified in both TBTO-treated and control cells; however, phosphorylation at residue S293, known to inhibit PDH activity, was identified only in control cells. A lower expression level of ribosomal protein S6 kinase 1, a downstream kinase of the mammalian target of rapamycin signaling pathway implicated in protein synthesis through phosphorylation of 40 ribosomal S6, was observed in the treated cells. Giant kinases like AMP-activated protein kinase (AMPK) and cAMP-dependent protein kinase (PKAR1A), which are known to mediate the phosphorylation of these enzymes, were identified in TBTO-treated cells. Downregulation of proteins, such as MAPK, matrin-3 and ribonucleotide reductase, subunit RRM2, which are implicated in cell proliferation, was also observed in TBTO-treated cells. Together, the results show that TBTO affects proliferation and energy sensor pathways.

A tale of two functions: enzymatic activity and translational repression by carboxyltransferase.

Acetyl-CoA Carboxylase catalyzes the first committed step in fatty acid synthesis. Escherichia coli acetyl-CoA carboxylase is composed of biotin carboxylase, carboxyltransferase and biotin carboxyl carrier protein functions. The accA and accD genes that code for the alpha- and beta-subunits, respectively, are not in an operon, yet yield an alpha(2)beta(2) carboxyltransferase. Here, we report that carboxyltransferase regulates its own translation by binding the mRNA encoding its subunits. This interaction is mediated by a zinc finger on the beta-subunit; mutation of the four cysteines to alanine diminished nucleic acid binding and catalytic activity. Carboxyltransferase binds the coding regions of both subunit mRNAs and inhibits translation, an inhibition that is relieved by the substrate acetyl-CoA. mRNA binding reciprocally inhibits catalytic activity. Preferential binding of carboxyltransferase to RNA in situ was shown using fluorescence resonance energy transfer. We propose an unusual regulatory mechanism by which carboxyltransferase acts as a 'dimmer switch' to regulate protein production and catalytic activity, while sensing the metabolic state of the cell through acetyl-CoA concentration.