Diazepam-binding inhibitor-related protein 1: a candidate autoantigen in acquired aplastic anemia patients harboring a minor population of paroxysmal nocturnal hemoglobinuria-type cells.
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To identify candidate antigens in aplastic anemia (AA), we screened proteins derived from a leukemia cell line with serum of an AA patient and identified diazepam-binding inhibitor-related protein 1 (DRS-1). Enzyme-linked immunosorbent assay (ELISA) revealed high titers of anti-DRS-1 antibodies (DRS-1 Abs) in 27 (38.0%) of 71 AA patients displaying increased paroxysmal nocturnal hemoglobinuria (PNH)-type cells (PNH(+)), 2 (6.3%) of 32 PNH(-) AA patients, 5 (38.5%) of 13 PNH(+) myelodysplastic syndrome (MDS) patients, and none of 42 PNH(-) MDS patients. DRS-1 gene was abundantly expressed in myeloid leukemia cell lines and in CD34(+) cells derived from healthy individuals. Stimulation of T cells from an AA patient displaying high DRS-1 Abs with a putative CD4(+) T-cell epitope (amino acid residues [aa's] 191-204) presented by HLA-DR15, which overlapped with a hot spot (aa's 173-198) of DRS-1 Ab epitopes, gave rise to T cells cytotoxic for L cells (murine fibroblasts) that were transfected with DRB1*1501 and DRS-1. Enzyme-linked immunospot assay demonstrated increased frequency of T-cell precursors specific to the DRS-1 peptide in other HLA-DR15(+) AA patients displaying high DRS-1 Ab titers. These findings indicate that DRS-1 may serve as an autoantigen eliciting immune attack against hematopoietic stem cells in a subset of AA patients characterized by increased PNH-type cells. (+info)
Isolation and characterization of a humoral factor that stimulates transcription of the acyl-CoA-binding protein in the pheromone gland of the silkmoth, Bombyx mori.
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Acyl-CoA binding protein (ACBP) is a highly conserved 10-kDa intracellular lipid-binding protein that binds straight-chain (C14-C22) acyl-CoA esters with high affinity and is expressed in a wide variety of species ranging from yeast to mammals. Functionally, ACBP can act as an acyl-CoA carrier or as an acyl-CoA pool maker within the cell. Much work on the biochemical properties regarding the ACBP has been performed using various vertebrate and plant tissues, as well as different types of cells in culture, the regulatory mechanisms underlying ACBP gene expression have remained poorly understood. By exploiting the unique sex pheromone production system in the moth pheromone gland (PG), we report that transcription of a specific ACBP termed pheromone gland ACBP is triggered by a hemolymph-based humoral factor. Following purification and structure elucidation by means of high resolution electrospray ionization mass spectrometry and NMR analyses, in conjunction with stereochemical analyses using acid hydrolysates, the humoral factor was identified to be beta-D-glucosyl-O-L-tyrosine. Examination of the hemolymph titers during development revealed that the amount of beta-D-glucosyl-O-L-tyrosine dramatically rose prior to eclosion and reached a maximum of 5 mg/ml (about 1 mg/pupa) on the day preceding eclosion, which was consistent with the effective dose of beta-D-glucosyl-O-L-tyrosine in stimulating pheromone gland ACBP transcription in vivo. Furthermore, in vitro assays using trimmed PG indicated that beta-D-glucosyl-O-L-tyrosine acts directly on the PG. These results provide the first evidence that transcription of some ACBPs can be triggered by specific humoral factors. (+info)
The gene encoding acyl-CoA-binding protein is subject to metabolic regulation by both sterol regulatory element-binding protein and peroxisome proliferator-activated receptor alpha in hepatocytes.
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The acyl-CoA-binding protein (ACBP) is a 10-kDa intracellular lipid-binding protein that transports acylCoA esters. The protein is expressed in most cell types at low levels; however, expression is particularly high in cells with a high turnover of fatty acids. Here we confirm a previous observation that ACBP expression in rodent liver is down-regulated by fasting, and we show that insulin but not glucose is the inducer of ACBP expression in primary rat hepatocytes. In keeping with the regulation by insulin, we show that ACBP is a sterol regulatory element-binding protein 1c (SREBP-1c) target gene in hepatocytes. Members of the SREBP family activate the rat ACBP gene through binding sites for SREBP and the auxiliary factors Sp1 and nuclear factor Y in the proximal promoter. In addition, we show that ACBP is a peroxisome proliferator-activated receptor (PPAR) alpha target gene in cultured hepatocytes and is induced in the liver by fibrates in a PPARalpha-dependent manner. Thus, ACBP is a dual PPARalpha and SREBP-1c target gene in hepatocytes. Fasting leads to reduced activity of SREBP but increased activity of PPARalpha in hepatocytes, and in keeping with ACBP being a dual target gene, we show that ACBP expression is significantly lower in livers from PPARalpha knock-out mice than in livers from wild type mice. In conclusion, expression of ACBP in rodent hepatocytes is subject to dual metabolic regulation by PPARalpha and SREBP-1c, which may reflect the need for ACBP during lipogenic as well as lipo-oxidative conditions. (+info)
Functional proteins involved in regulation of intracellular Ca(2+) for drug development: chronic nicotine treatment upregulates L-type high voltage-gated calcium channels.
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Neurochemical mechanisms underlying drug dependence and withdrawal syndrome remain unclear. In this review, we discuss how chronic nicotine exposure to neurons affects expression of diazepam binding inhibitor (DBI), an endogenous anxiogenic neuropeptide supposed to be a common substance participating drug dependence, and function of L-type high voltage-gated Ca(2+) channels (HVCCs). We also discuss the functional interaction between DBI and L-type HVCCs in nicotine dependence. Both DBI levels and [(45)Ca(2+)] influx significantly increased in the brain from mice treated with nicotine for long term, which was further enhanced after abrupt cessation of nicotine and was abolished by nicotinic acetylcholine receptor (nAChR) antagonists. Similar responses of DBI expression and L-type HVCC function were observed in cerebral cortical neurons after sustained exposure to nicotine. In addition, increased DBI expression was inhibited by antagonists of nAChR and L-type HVCCs. Sustained exposure of neurons to nicotine significantly enhanced expression of alpha(1) and alpha(2)/delta(1) subunits for L-type HVCCs and caused an increase in the B(max) value of [(3)H]verapamil binding to the particulate fractions. Therefore, it is concluded that the alterations in DBI expression is mediated via increased influx of Ca(2+) through upregulated L-type HVCCs and these neurochemical changes have a close relationship with development of nicotine dependence and/or its withdrawal syndrome. (+info)
Peptide signaling during terminal differentiation of Dictyostelium.
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A wide variety of mechanisms have evolved for intercellular communication in metazoans, but some of the signaling molecules were already used in their predecessors. The social amoeba, Dictyostelium discoideum, is known to use peptides to trigger sporulation within fruiting bodies, but their sequences have not been defined. We found that the peptide signal spore differentiation factor 2 (SDF-2) is processed from acyl-CoA binding protein, AcbA. The mammalian homolog of AcbA is processed to diazepam binding inhibitor that binds to the GABA(A) receptor in the brain and to peripheral 1,4 benzodiazepine receptors. Although Dictyostelium has neither GABA(A) nor peripheral-type benzodiazepine receptors, we find that both a diazepam binding inhibitor peptide and diazepam (Valium) can mimic SDF-2 in a Dictyostelium bioassay. Mutants lacking AcbA sporulate well only when developed in chimeras with WT cells. Using a yeast system we show that ligand binding to the SDF-2 receptor histidine kinase, DhkA, inhibits phosphorelay, which can account for its ability to induce rapid sporulation. (+info)
Regulation of lipolytic activity by long-chain acyl-coenzyme A in islets and adipocytes.
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Intracellular lipolysis is a major pathway of lipid metabolism that has roles, not only in the provision of free fatty acids as energy substrate, but also in intracellular signal transduction. The latter is likely to be particularly important in the regulation of insulin secretion from islet beta-cells. The mechanisms by which lipolysis is regulated in different tissues is, therefore, of considerable interest. Here, the effects of long-chain acyl-CoA esters (LC-CoA) on lipase activity in islets and adipocytes were compared. Palmitoyl-CoA (Pal-CoA, 1-10 microM) stimulated lipase activity in islets from both normal and hormone-sensitive lipase (HSL)-null mice and in phosphatase-treated islets, indicating that the stimulatory effect was neither on HSL nor phosphorylation dependent. In contrast, we reproduced the previously published observations showing inhibition of HSL activity by LC-CoA in adipocytes. The inhibitory effect of LC-CoA on adipocyte HSL was dependent on phosphorylation and enhanced by acyl-CoA-binding protein (ACBP). In contrast, the stimulatory effect on islet lipase activity was blocked by ACBP, presumably due to binding and sequestration of LC-CoA. These data suggest the following intertissue relationship between islets and adipocytes with respect to fatty acid metabolism, LC-CoA signaling, and lipolysis. Elevated LC-CoA in islets stimulates lipolysis to generate a signal to increase insulin secretion, whereas elevated LC-CoA in adipocytes inhibits lipolysis. Together, these opposite actions of LC-CoA lower circulating fat by inhibiting its release from adipocytes and promoting fat storage via insulin action. (+info)
Purification and characterization of variants of acyl-CoA-binding protein in the bovine liver.
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Four differently modified forms of acyl-CoA-binding protein (ACBP) were identified in ACBP purified from bovine liver. The majority of the purified ACBP was focused at pH 5.9 in isoelectric focusing and could be shown to be N-acetylated ACBP without any further modifications. Two minor peaks were focused at pH 5.25 and 4.85 respectively. Mass spectrometry and sequence determination showed that the pI 5.25 form was acetylated at Lys18 and that the pI 4.85 form was malonylated in the same position. Furthermore, it could be shown that non-enzymic glycosylation occurred during purification. The acetylated and malonylated variants of ACBP were only found in adult cattle. (+info)
Probing structural heterogeneities and fluctuations of nucleic acids and denatured proteins.
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We study protein and nucleic acid structure and dynamics using single-molecule FRET and alternating-laser excitation. Freely diffusing molecules are sorted into subpopulations based on single-molecule signals detected within 100 micros to 1 ms. Distance distributions caused by fluctuations faster than 100 micros are studied within these subpopulations by using time-correlated single-photon counting. Measured distance distributions for dsDNA can be accounted for by considering fluorophore linkers and fluorophore rotational diffusion, except that we find smaller fluctuations for internally labeled dsDNA than DNA with one of the fluorophores positioned at a terminal site. We find that the electrostatic portion of the persistence length of short single-stranded poly(dT) varies approximately as the ionic strength (I) to the -1/2 power (I(-1/2)), and that the average contribution to the contour length per base is 0.40-0.45 nm. We study unfolded chymotrypsin inhibitor 2 (CI2) and unfolded acyl-CoA binding protein (ACBP) even under conditions where folded and unfolded subpopulations coexist (contributions from folded proteins are excluded by using alternating-laser excitation). At lower denaturant concentrations, unfolded CI2 and ACBP are more compact and display larger fluctuations than at higher denaturant concentrations where only unfolded proteins are present. The experimentally measured fluctuations are larger than the fluctuations predicted from a Gaussian chain model or a wormlike chain model. We propose that the larger fluctuations may indicate transient residual structure in the unfolded state. (+info)