scholarly journals Expression, purification and functional characterization of recombinant human acyl-CoA-binding protein (ACBP) from erythroid cells.

2010 ◽  
Vol 57 (4) ◽  
Author(s):  
Katarzyna Augoff ◽  
Adam Kolondra ◽  
Anna Chorzalska ◽  
Agnieszka Lach ◽  
Krzysztof Grabowski ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Functional Characterization ◽  
Fatty Acyl ◽  
Covalent Attachment ◽  
Erythroid Cells ◽  
Dynamic Regulation ◽  
Protein Palmitoylation ◽  
Chain Acyl ◽  
Acyl Coenzyme A

Fatty acyl-CoA esters are extremely important in cellular homeostasis. They are intermediates in both lipid metabolism and post-translational protein modifications. Among these modification events, protein palmitoylation seems to be unique by its reversibility which allows dynamic regulation of the protein hydrophobicity. The recent discovery of an enzyme family that catalyze protein palmitoylation has increased the understanding of the enzymology of the covalent attachment of fatty acids to proteins. Despite that, the molecular mechanism of supplying acyl-CoA esters to this reaction is yet to be established. Acyl-coenzyme A-binding proteins are known to bind long-chain acyl-CoA esters with very high affinity. Therefore, they play a significant role in intracellular acyl-CoA transport and pool formation. The purpose of this work is to explore the potential of one of the acyl-CoA-binding proteins to participate in the protein palmitoylation. In this study, a recombinant form of ACBP derived from human erythroid cells was expressed in E. coli, purified, and functionally characterized. We demonstrate that recombinant hACBP effectively binds palmitoyl-CoA in vitro, undergoing a shift from a monomeric to a dimeric state, and that this ligand-binding ability is involved in erythrocytic membrane phosphatidylcholine (PC) remodeling but not in protein acylation.

scholarly journals Amelioration of Cryptosporidium parvum Infection In Vitro and In Vivo by Targeting Parasite Fatty Acyl-Coenzyme A Synthetases

2013 ◽  
Vol 209 (8) ◽  
pp. 1279-1287 ◽  
Author(s):  
F. Guo ◽  
H. Zhang ◽  
J. M. Fritzler ◽  
S. D. Rider ◽  
L. Xiang ◽  
...  
Keyword(s):  
Cryptosporidium Parvum ◽  
Coenzyme A ◽  
Fatty Acyl ◽  
Acyl Coenzyme A

scholarly journals Rescue of GATA-1-deficient embryonic stem cells by heterologous GATA-binding proteins.

1995 ◽  
Vol 15 (2) ◽  
pp. 626-633 ◽  
Author(s):  
G A Blobel ◽  
M C Simon ◽  
S H Orkin
Keyword(s):  
Zinc Finger ◽  
Binding Proteins ◽  
Es Cells ◽  
Zinc Fingers ◽  
Embryonic Stem ◽  
Transcription Activation ◽  
Erythroid Cells ◽  
Zinc Finger Region

Totipotent murine embryonic stem (ES) cells can be differentiated in vitro to form embryoid bodies (EBs) containing hematopoietic cells of multiple lineages, including erythroid cells. In vitro erythroid development parallels that which is observed in vivo. ES cells in which the gene for the erythroid transcription factor GATA-1 has been disrupted fail to produce mature erythroid cells either in vivo or in vitro. With the EB in vitro differentiation assay, constructs expressing heterologous GATA-binding proteins were tested for their abilities to correct the developmental defect of GATA-1-deficient ES cells. The results presented here show that the highly divergent chicken GATA-1 can rescue GATA-1 deficiency to an extent similar to that of murine GATA-1 (mGATA-1), as determined by size and morphology of EBs, presence of red cells, and globin gene expression. Furthermore, GATA-3 and GATA-4, which are normally expressed in different tissues, and a protein consisting of the zinc fingers of GATA-1 fused to the herpes simplex virus VP16 transcription activation domain were able to compensate for the GATA-1 defect. Chimeric molecules in which both zinc fingers of mGATA-1 were replaced with the zinc fingers of human GATA-3 or with the single finger of the fungal GATA factor areA, as well as a construct bearing the zinc finger region alone, displayed rescue activity. These results suggest that neither the transcription activation domains of mGATA-1 nor its zinc fingers impart erythroid cell specificity for its action in vivo. Rather, it appears that specificity is mediated through the cis-acting control regions which determine spatial and temporal expression of the GATA-1 gene. Furthermore, our results demonstrate that the zinc finger region may have a biological function in addition to mediating DNA binding.

scholarly journals A novel cycle involving fatty acyl-coenzyme A regulates asialoglycoprotein receptor activity in permeable hepatocytes.

1994 ◽  
Vol 5 (2) ◽  
pp. 227-235 ◽  
Author(s):  
P H Weigel ◽  
J D Medh ◽  
J A Oka
Keyword(s):  
Rat Hepatocytes ◽  
Fatty Acyl ◽  
Binding Activity ◽  
Asialoglycoprotein Receptor ◽  
Receptor Activity ◽  
Independent Effect ◽  
Palmitoyl Carnitine ◽  
Acyl Coenzyme A ◽  
Chain Lengths

Asialoglycoprotein receptors (ASGP-Rs) in permeable rat hepatocytes can be inactivated in the absence of ligand. This cytosol-independent effect is relatively slow (t1/2 approximately 12 min) and is temperature and ATP dependent. Here we show that in the absence of cytosol, the addition of palmitoyl-CoA (Pal-CoA) rapidly (t1/2 < 0.4 min) and quantitatively reactivates the inactivated receptors. Receptor reactivation was half-maximal at approximately 10-12 microM free Pal-CoA at 37 degrees C. Although substantially higher total concentrations were used, much of the added Pal-CoA was cell associated and not free. The effects of Pal-CoA were eliminated by bovine serum albumin at concentrations sufficient to bind all free monomeric fatty acyl-CoA, suggesting that micellar effects are not responsible for the ability to reactivate ASGP-Rs. Also, palmitoyl-carnitine did not substitute for Pal-CoA. The initial ASGP-R inactivation is not affected by treating cells with N-ethylmaleimide or by a KCl wash but is inhibited by sodium orthovanadate or high Ca2+ levels. Myristoyl-CoA (C14) was also able to reactivate inactive ASGP-Rs about as well as Pal-CoA. Fatty acyl-CoAs with chain lengths of C12 (lauroyl) or C18 (steroyl) were < 50% as active. The ligand binding activity of these receptors can subsequently be modulated within minutes by the further addition of ATP or Pal-CoA to achieve additional rounds of ASGP-R inactivation or reactivation, respectively. These in vitro data demonstrate the occurrence of a novel asialoglycoprotein receptor inactivation-reactivation cycle that could regulate receptor activity during endocytosis and receptor recycling.

scholarly journals Acyl-Coenzyme A Dehydrogenases Are Localized on GLUT4-Containing Vesicles via Association with Insulin-Regulated Aminopeptidase in a Manner Dependent on Its Dileucine Motif

2002 ◽  
Vol 16 (5) ◽  
pp. 1049-1059 ◽  
Author(s):  
Hideki Katagiri ◽  
Tomoichiro Asano ◽  
Tetsuya Yamada ◽  
Toshifumi Aoyama ◽  
Yasushi Fukushima ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Coenzyme A ◽  
Amino Acid Residues ◽  
Glutathione S Transferase ◽  
Medium Chain ◽  
Dileucine Motif ◽  
Chain Acyl ◽  
Acyl Coenzyme A ◽  
Long Chain

Abstract Insulin-regulated aminopeptidase (IRAP, also termed vp165) is known to be localized on the GLUT4-containing vesicles and to be recruited to the plasma membrane after stimulation with insulin. The cytoplasmic region of IRAP contains two dileucine motifs and acidic regions, one of which (amino acid residues 55–82) is reportedly involved in retention of GLUT4-containing vesicles. The region of IRAP fused with glutathione-S-transferase [GST-IRAP(55–82)] was incubated with lysates from 3T3-L1 adipocytes, leading to identification of long-chain, medium-chain, and short-chain acyl-coenzyme A dehydrogenases (ACDs) as the proteins associated with IRAP. The association was nearly abolished by mutation of the dileucine motif of IRAP. Immunoblotting of fractions prepared from sucrose gradient ultracentrifugation and vesicles immunopurified with anti-GLUT4 antibody revealed these ACDs to be localized on GLUT4-containing vesicles. Furthermore, 3-mercaptopropionic acid and hexanoyl-CoA, inhibitors of long-chain and medium-chain ACDs, respectively, induced dissociation of long-chain acyl-coenzyme A dehydrogenase and/or medium-chain acyl-coenzyme A dehydrogenase from IRAP in vitro as well as recruitment of GLUT4 to the plasma membrane and stimulation of glucose transport activity in permeabilized 3T3-L1 adipocytes. These findings suggest that ACDs are localized on GLUT4-containing vesicles via association with IRAP in a manner dependent on its dileucine motif and play a role in retention of GLUT4-containing vesicles to an intracellular compartment.

scholarly journals Regulatory effects of fatty acyl-coenzyme A derivatives on phosphate-activated pig brain and kidney glutaminase in vitro

1975 ◽  
Vol 149 (1) ◽  
pp. 83-91 ◽  
Author(s):  
E Kvamme ◽  
I A Torgner
Keyword(s):  
Acyl Chain ◽  
Bromothymol Blue ◽  
Fatty Acyl ◽  
Fatty Acyl Chain ◽  
Pig Brain ◽  
Low Concentrations ◽  
Regulatory Effects ◽  
Acyl Coenzyme A ◽  
Carnitine Derivatives

1. Fatty n-acyl-CoA derivatives in the concentration range 5 μM-0.1mM and with 5-18 fatty acyl carbons have dual effects on phosphate-activated glutaminase from pig brain and kidney. Generally, fatty acyl-CoA derivatives in low concentrations activate the enzyme, but inhibit at higher concentrations; phosphate and citrate potentiate the activation, displaying positive co-operatively, and protect against inactivation. The fatty acyl-CoA derivatives affect glutaminase similarly to Bromothymol Blue, but differently from acetyl-CoA, which activates the enzyme only at very low phosphate or citrate concentrations. 2. Saturated fatty acyl-CoA derivatives, with 5-10 fatty acyl carbons, only activate the enzyme in the concentration range 0-0.1 mM. When the fatty acyl chain is elongated, the fatty acyl-CoA derivatives gradually become more powerful inhibitors of glutaminase at the expense of their activating capacity. In particular, palmitoyl-CoA and stearoyl-CoA are strong inhibitors at concentrations (10 μM) at which the corresponding free fatty acids and fatty acyl-carnitine derivatives have no effect. 3. The unsaturated fatty acyl-CoA derivatives, oleoyl-CoA and linoleoyl-CoA, behave as potent activators in the lower part of the concentration range tested (0-0.05mM), and as inhibitors in the upper part of this range (0.02-0.10mM). Oleic acid and linoleic acid have similar properties, but their activating capacity is less pronounced. 4. Phosphate both prevented and reversed the inhibition, but no restoration of activity was possible once the enzyme became inactivated. 5. By changing the pH from 7.0 to 8.0 the activating capacity of the fatty acyl-CoA derivatives is increased, as is their concentration range for activation. 6. The fatty acyl-CoA derivatives are somewhat more potent activator for brain glutaminase, but otherwise they affect the two enzymes similarly.

LIPID SYNTHESIS IN RAT LIVER PARTICLES: DIVERSION OF SYNTHESIS FROM TRIGLYCERIDE TO LECITHIN BY THE IN VITRO ADDITION OF CYTIDINE DIPHOSPHATE CHOLINE

1962 ◽  
Vol 40 (1) ◽  
pp. 1051-1058 ◽  
Author(s):  
K. P. Strickland ◽  
R. J. Rossiter
Keyword(s):  
Fatty Acid ◽  
Phosphatidic Acid ◽  
Experimental Test ◽  
Lipid Synthesis ◽  
Phosphatidyl Inositol ◽  
Fatty Acyl ◽  
Phosphatidic Acids ◽  
Acyl Coenzyme A ◽  
Cytidine Diphosphate

When particle preparations from rat or chicken liver were incubated in a suitable medium containing α-glycerophosphate-C14, radioactivity was recovered from lecithin, phosphatidyl inositol, phosphatidic acid, and triglyceride. Whole homogenate and various particle preparations catalyzed the dephosphorylation of a number of phosphatidic acids, with the liberation of inorganic P.It is currently believed that liver preparations are capable of catalyzing the esterification of L-α-glycerophosphate, with the formation of L-α-phosphatidic acid, which subsequently may be dephosphorylated to form D-α,β-diglyceride. The diglyceride so formed may then give rise either to lecithin, by combining with phosphorylcholine from cytidine diphosphate choline, or to triglyceride, by combining with fatty acid from fatty acyl coenzyme A. If these reactions occur in liver particle preparations, it should be possible, by the addition in vitro of unlabelled cytidine diphosphate choline, to divert the synthesis of lipid from the formation of triglyceride to the formation of lecithin. In experiments designed to put this hypothesis to the experimental test, such a diversion of lipid synthesis was achieved.

scholarly journals Transcriptional Regulation by the Short-Chain Fatty Acyl Coenzyme A Regulator (ScfR) PccR Controls Propionyl Coenzyme A Assimilation by Rhodobacter sphaeroides

2015 ◽  
Vol 197 (19) ◽  
pp. 3048-3056 ◽  
Author(s):  
Michael S. Carter ◽  
Birgit E. Alber
Keyword(s):  
Rhodobacter Sphaeroides ◽  
Coenzyme A ◽  
Bacterial Species ◽  
Fatty Acyl ◽  
Short Chain ◽  
Coding Region ◽  
Acetyl Coa ◽  
Content Type ◽  
Chain Acyl ◽  
Acyl Coenzyme A

ABSTRACTPropionyl coenzyme A (propionyl-CoA) assimilation byRhodobacter sphaeroidesproceeds via the methylmalonyl-CoA pathway. The activity of the key enzyme of the pathway, propionyl-CoA carboxylase (PCC), was upregulated 20-fold during growth with propionate compared to growth with succinate. Because propionyl-CoA is an intermediate in acetyl-CoA assimilation via the ethylmalonyl-CoA pathway, acetate growth also requires the methylmalonyl-CoA pathway. PCC activities were upregulated 8-fold in extracts of acetate-grown cells compared to extracts of succinate-grown cells. The upregulation of PCC activities during growth with propionate or acetate corresponded to increased expression of thepccBgene, which encodes a subunit of PCC. PccR (RSP_2186) was identified to be a transcriptional regulator required for the upregulation ofpccBtranscript levels and, consequently, PCC activity: growth substrate-dependent regulation was lost whenpccRwas inactivated by an in-frame deletion. In thepccRmutant,lacZexpression from a 215-bp plasmid-bornepccBupstream fragment including 27 bp of thepccBcoding region was also deregulated. A loss of regulation as a result of mutations in the conserved motifs TTTGCAAA-X4-TTTGCAAA in the presence of PccR allowed the prediction of a possible operator site. PccR, together with homologs from other organisms, formed a distinct clade within the family ofshort-chainfatty acyl coenzyme Aregulators (ScfRs) defined here. Some members from other clades within the ScfR family have previously been shown to be involved in regulating acetyl-CoA assimilation by the glyoxylate bypass (RamB) or propionyl-CoA assimilation by the methylcitrate cycle (MccR).IMPORTANCEShort-chain acyl-CoAs are intermediates in essential biosynthetic and degradative pathways. The regulation of their accumulation is crucial for appropriate cellular function. This work identifies a regulator (PccR) that prevents the accumulation of propionyl-CoA by controlling expression of the gene encoding propionyl-CoA carboxylase, which is responsible for propionyl-CoA consumption byRhodobacter sphaeroides. Many otherProteobacteriaandActinomycetalescontain one or several PccR homologs that group into distinct clades on the basis of the pathway of acyl-CoA metabolism that they control. Furthermore, an upstream analysis of genes encoding PccR homologs allows the prediction of conserved binding motifs for these regulators. Overall, this study evaluates a single regulator of propionyl-CoA assimilation while expanding the knowledge of the regulation of short-chain acyl-CoAs in many bacterial species.

scholarly journals Role of long-chain fatty acyl-CoA esters in the regulation of metabolism and in cell signalling

1997 ◽  
Vol 323 (1) ◽  
pp. 1-12 ◽  
Author(s):  
Nils Joakim FÆRGEMAN ◽  
Jens KNUDSEN
Keyword(s):  
Binding Proteins ◽  
Feedback Inhibition ◽  
Fatty Acyl ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Release Channel ◽  
Δ9 Desaturase ◽  
Chain Acyl ◽  
Long Chain

The intracellular concentration of free unbound acyl-CoA esters is tightly controlled by feedback inhibition of the acyl-CoA synthetase and is buffered by specific acyl-CoA binding proteins. Excessive increases in the concentration are expected to be prevented by conversion into acylcarnitines or by hydrolysis by acyl-CoA hydrolases. Under normal physiological conditions the free cytosolic concentration of acyl-CoA esters will be in the low nanomolar range, and it is unlikely to exceed 200 nM under the most extreme conditions. The fact that acetyl-CoA carboxylase is active during fatty acid synthesis (Ki for acyl-CoA is 5 nM) indicates strongly that the free cytosolic acyl-CoA concentration is below 5 nM under these conditions. Only a limited number of the reported experiments on the effects of acyl-CoA on cellular functions and enzymes have been carried out at low physiological concentrations in the presence of the appropriate acyl-CoA-buffering binding proteins. Re-evaluation of many of the reported effects is therefore urgently required. However, the observations that the ryanodine-senstitive Ca2+-release channel is regulated by long-chain acyl-CoA esters in the presence of a molar excess of acyl-CoA binding protein and that acetyl-CoA carboxylase, the AMP kinase kinase and the Escherichia coli transcription factor FadR are affected by low nanomolar concentrations of acyl-CoA indicate that long-chain acyl-CoA esters can act as regulatory molecules in vivo. This view is further supported by the observation that fatty acids do not repress expression of acetyl-CoA carboxylase or Δ9-desaturase in yeast deficient in acyl-CoA synthetase.

scholarly journals Differences in Substrate Specificities of Five Bacterial Wax Ester Synthases

2012 ◽  
Vol 78 (16) ◽  
pp. 5734-5745 ◽  
Author(s):  
Brett M. Barney ◽  
Bradley D. Wahlen ◽  
EmmaLee Garner ◽  
Jiashi Wei ◽  
Lance C. Seefeldt
Keyword(s):  
Kinetic Studies ◽  
Fatty Acyl ◽  
Biofuel Production ◽  
Wax Ester ◽  
Acinetobacter Baylyi ◽  
Content Type ◽  
Rapid Purification ◽  
Range Characterization ◽  
Acyl Coenzyme A

ABSTRACTWax esters are produced in certain bacteria as a potential carbon and energy storage compound. The final enzyme in the biosynthetic pathway responsible for wax ester production is the bifunctional wax ester synthase/acyl-coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT), which utilizes a range of fatty alcohols and fatty acyl-CoAs to synthesize the corresponding wax ester. We report here the isolation and substrate range characterization for five WS/DGAT enzymes from four different bacteria:Marinobacter aquaeoleiVT8,Acinetobacter baylyi,Rhodococcus jostiiRHA1, andPsychrobacter cryohalolentisK5. The results from kinetic studies of isolated enzymes reveal a differential activity based on the order of substrate addition and reveal subtle differences between the substrate selectivity of the different enzymes. Thesein vitroresults are compared to the wax ester and triacylglyceride product profiles obtained from each organism grown under neutral lipid accumulating conditions, providing potential insights into the role that the WS/DGAT enzyme plays in determining the final wax ester products that are produced under conditions of nutrient stress in each of these bacteria. Further, the analysis revealed that one enzyme in particular fromM. aquaeoleiVT8 showed the greatest potential for future study based on rapid purification and significantly higher activity than was found for the other isolated WS/DGAT enzymes. The results provide a framework to test prospective differences between these enzymes for potential biotechnological applications such as high-value petrochemicals and biofuel production.

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