Ascaridia galli fatty acid-binding protein, a member of the nematode polyprotein allergens family.

A fatty acid-binding protein from the nematode Ascaridia galli was characterized. The gene was isolated and recombinantly expressed in Escherichia coli. According to the deduced amino acid sequence A. galli fatty acid-binding protein (AgFABP) belongs to the family of nematode polyprotein allergens, as shown by Western blotting and PCR analysis with genomic DNA and cDNA. Both native and recombinant proteins bind fatty acids and retinoids with high affinity. The fluorescent fatty acid analogue 11-[(5-dimethylaminonaphthalene-1-sulfonyl)amino] undecanoic acid (DAUDA) shows substantial changes in its emission spectrum when bound to AgFABP; this binding is reversed by fatty acids such as oleate. Moreover, changes of the intrinsic fluorescence of retinol and retinoic acid confirm retinoid binding activity of AgFABP. Fluorescence titration experiments with DAUDA indicate stoichiometric binding to a single binding site per monomer unit with affinities (Kd) of 1.6 and 1.8 x 10(-7) m for native and the recombinant protein, respectively. The apparent binding affinities of the nonfluorescent ligands were calculated in displacement experiments with DAUDA and values in the same range were obtained for myristic, palmitic, oleic, linoleic, arachidonic and retinoic acid. Additionally, the binding affinity of AgFABP for oleate and palmitate was determined by direct and indirect radiochemical analysis and the values obtained were similar to those from the fluorescent experiments. Both proteins show a preference for the binding of long-chain saturated and unsaturated fatty acids, but not for short chain (C3-C12) and branched fatty acids, cholesterol and tryptophan.     

Characterization of a novel member of the DOK family that binds and modulates Abl signaling

A novel member of the p62(dok) family of proteins, termed DOKL, is described. DOKL contains features of intracellular signaling molecules, including an N-terminal PH (pleckstrin homology) domain, a central PTB (phosphotyrosine binding) domain, and a C-terminal domain with multiple potential tyrosine phosphorylation sites and proline-rich regions, which might serve as docking sites for SH2- and SH3-containing proteins. The DOKL gene is predominantly expressed in bone marrow, spleen, and lung, although low-level expression of the RNA can also be detected in other tissues. DOKL and p62(dok) bind through their PTB domains to the Abelson tyrosine kinase in a kinase-dependent manner in both yeast and mammalian cells. DOKL is phosphorylated by the Abl tyrosine kinase in vivo. In contrast to p62(dok), DOKL lacks YxxP motifs in the C terminus and does not bind to Ras GTPase-activating protein (RasGAP) upon phosphorylation. Overexpression of DOKL, but not p62(dok), suppresses v-Abl-induced mitogen-activated protein (MAP) kinase activation but has no effect on constitutively activated Ras- and epidermal growth factor-induced MAP kinase activation. The inhibitory effect requires the PTB domain of DOKL. Finally, overexpression of DOKL in NIH 3T3 cells inhibits the transforming activity of v-Abl. These results suggest that DOKL may modulate Abl function.     

Characterization of a fatty acid-binding protein from rat heart

A fatty acid-binding protein has been isolated from rat heart and purified by gel filtration chromatography on Sephadex G-75 and anion-exchange chromatography on DE52. The circular dichroic spectrum of this protein was not affected by protein concentration, suggesting that it does not aggregate into multimers. Computer analyses of the circular dichroic spectrum predicted that rat heart fatty acid-binding protein contains approximately 22% alpha-helix, 45% beta-form and 33% unordered structure. Immunological studies showed that the fatty acid-binding proteins from rat heart and rat liver are immunochemically unrelated. The amino acid composition and partial amino acid sequence of the heart protein indicated that it is structurally related to, but distinct from, other fatty acid-binding proteins from liver, intestine, and 3T3 adipocytes. Using a binding assay which measures the transfer of fatty acids between donor liposomes and protein (Brecher, P., Saouaf, R., Sugarman, J. M., Eisenberg, D., and LaRosa, K. (1984) J. Biol. Chem. 259, 13395-13401), it was shown that both rat heart and liver fatty acid-binding proteins bind 2 mol of oleic acid or palmitic acid/mol of protein. The structural and functional relationship of rat heart fatty acid-binding protein to fatty acid-binding proteins from other tissues is discussed.     

The adipocyte fatty acid-binding protein binds to membranes by electrostatic interactions

The adipocyte fatty acid-binding protein (AFABP) is believed to transfer unesterified fatty acids (FA) to phospholipid membranes via a collisional mechanism that involves ionic interactions between lysine residues on the protein surface and phospholipid headgroups. This hypothesis is derived largely from kinetic analysis of FA transfer from AFABP to membranes. In this study, we examined directly the binding of AFABP to large unilamellar vesicles (LUV) of differing phospholipid compositions. AFABP bound LUV containing either cardiolipin or phosphatidic acid, and the amount of protein bound depended upon the mol % anionic phospholipid. The K(a) for CL or PA in LUV containing 25 mol % of these anionic phospholipids was approximately 2 x 10(3) M(-1). No detectable binding occurred when AFABP was mixed with zwitterionic membranes, nor when acetylated AFABP in which surface lysines had been chemically neutralized was mixed with anionic membranes. The binding of AFABP to acidic membranes depended upon the ionic strength of the incubation buffer: >/=200 mM NaCl reduced protein-lipid complex formation in parallel with a decrease in the rate of FA transfer from AFABP to negatively charged membranes. It was further found that AFABP, but not acetylated AFABP, prevented cytochrome c, a well characterized peripheral membrane protein, from binding to membranes. These results directly demonstrate that AFABP binds to anionic phospholipid membranes and suggest that, although generally described as a cytosolic protein, AFABP may behave as a peripheral membrane protein to help target fatty acids to and/or from intracellular sites of utilization.     

Muscle fatty acid-binding protein

Muscle or heart fatty acid-binding protein is a low molecular weight protein that binds long-chain fatty acids in the cytosol of muscle tissues. The three-dimensional structure of the human, bovine and insect proteins are known, either via X-ray or NMR techniques. The folding of the protein closely resembles that of the other FABPs: ten anti-parallel beta-strands are arranged to form a clam shell, closed at one end by two alpha-helices. This arrangement allows the formation of an internal cavity where the fatty acid can be accommodated, protected and isolated from the external environment. The fatty acid in the protein interior is stabilized by electrostatic and hydrogen bond interactions of its carboxylic head with charged or polar residues of the protein and by interactions of its tail with hydrophobic residues. The three-dimensional structure of different fatty acid-protein complexes along with molecular dynamics simulations are now providing insight into the molecular details of the specificity of the ligand binding.     

A new member of the LIM protein family binds to filamin B and localizes at stress fibers

Human filamins are 280-kDa proteins containing an N-terminal actin-binding domain followed by 24 characteristic repeats. They also interact with a number of other cellular proteins. All of those identified to date, with the exception of actin, bind to the C-terminal third of a filamin. In a yeast two-hybrid search of a human placental library, using as bait repeats 10-18 of filamin B, we isolated a cDNA coding for a novel 374 amino acid protein containing a proline-rich domain near its N terminus and two LIM domains at its C terminus. We term this protein filamin-binding LIM protein-1, FBLP-1. Yeast two-hybrid studies with deletion mutants localized the areas of interaction in FBLP-1 to its N-terminal domain and in filamin B to repeats 10-13. FBLP-1 mRNA was detected in a variety of tissues and cells including platelets and endothelial cells. We also have identified two FBLP-1 variants. Both contain three C-terminal LIM domains, but one lacks the N-terminal proline-rich domain. Transfection of FBLP-1 into 293A cells promoted stress fiber formation, and both FBLP-1 and filamin B localized to stress fibers in the transfected cells. The association between filamin B and FBLP-1 may play a hitherto unknown role in cytoskeletal function, cell adhesion, and cell motility.     

Myosin phosphatase-Rho interacting protein. A new member of the myosin phosphatase complex that directly binds RhoA

Regulation of vascular smooth muscle cell contractile state is critical for the maintenance of blood vessel tone. Abnormal vascular smooth muscle cell contractility plays an important role in the pathogenesis of hypertension, blood vessel spasm, and atherosclerosis. Myosin phosphatase, the key enzyme controlling myosin light chain dephosphorylation, regulates smooth muscle cell contraction. Vasoconstrictor and vasodilator pathways inhibit and activate myosin phosphatase, respectively. G-protein-coupled receptor agonists can inhibit myosin phosphatase and cause smooth muscle cell contraction by activating RhoA/Rho kinase, whereas NO/cGMP can activate myosin phosphatase and cause smooth muscle cell relaxation by activation of cGMP-dependent protein kinase. We have used yeast two-hybrid screening to identify a 116-kDa human protein that interacts with both myosin phosphatase and RhoA. This myosin phosphatase-RhoA interacting protein, or M-RIP, is highly homologous to murine p116RIP3, is expressed in vascular smooth muscle, and is localized to actin myofilaments. M-RIP binds directly to the myosin binding subunit of myosin phosphatase in vivo in vascular smooth muscle cells by an interaction between coiled-coil and leucine zipper domains in the two proteins. An adjacent domain of M-RIP directly binds RhoA in a nucleotide-independent manner. M-RIP copurifies with RhoA and Rho kinase, colocalizes on actin stress fibers with RhoA and MBS, and is associated with Rho kinase activity in vascular smooth muscle cells. M-RIP can assemble a complex containing both RhoA and MBS, suggesting that M-RIP may play a role in myosin phosphatase regulation by RhoA.     

Cerato-platanin, the first member of a new fungal protein family

The ascomcete Ceratocystis fimbriata, the causal agent of “canker stain disease,” secretes a protein of 12.4 kDa that elicits phytoalexin synthesis and plant cell death. This protein, named cerato-platanin (CP), is also located in the cell walls of ascospores, hyphae, and conidia; it contains four cysteines (S-S bridged) and is moderately hydrophobic. The cp gene consists of a single exon and has 42 bp codifying for a signal peptide of 14 residues. The recombinant protein was obtained by cloning the cp gene of the mature protein in Escherichia coli (BL21), and a refolding step was needed to achieve the native active form. In the European Molecular Biology data bank, CP is reported as the first member of the CP family; this is the first example of an set of secreted fungal proteins whose primary structure is very similar. Nonetheless, the data also revealed some structural and functional features that make CP simlar to proteins of the hydrophobin family.     

Heme-binding by Drosophila retinoid- and fatty acid-binding glycoprotein (RFABG), a member of the proapolipophorin gene family.

We previously have cloned and characterized a retinoid- and fatty acid-binding glycoprotein (RFABG) isolated from the heads of Drosophila melanogaster. The protein is composed of two glycosylated subunits (Mr = >200,000 and 70,000) and is a member of the proapolipophorin gene family. Spectral analysis of purified RFABG revealed an absolute absorbance peak at 405 nm, which is typical for a heme-containing protein. The aim of the present study was to characterize the heme-binding properties of RFABG. Upon saturation of the protein solution with carbon monoxide followed by dithionite reduction, a red shift of the Soret peak to 424 nm and the characteristic alpha- and beta- bands at 567 and 539 nm were observed. Native RFABG contains approximately 0.175 moles of heme (mol/mol) indicating that purified RFABG is primarily the apoprotein. Hemin-agarose affinity chromatography of the native RFABG followed by Western blot analysis showed a single immunoreactive band at 70 kDa, indicating that the heme-binding domain resides in the 70 kDa subunit. Although retinoid and fatty acid also bind to the 70 kDa subunit, no competition was observed when an excess of heme was added to a solution of retinoid or fatty acid bound to RFABG. Heme added to a solution of purified RFABG bound in a saturable manner with an affinity of 3.8 x 10(-7) m.Thus, the current study clearly demonstrates that retinoid- and fatty acid-binding glycoprotein is a novel heme-binding protein, which may be involved in the transport and/or metabolism of heme in Drosophila.     

脂肪酸结合蛋白的研究进展

脂脉酸结合蛋白（ＦＡＢＰ）是一族小分子细胞内蛋白质,对长链脂肪酸有很高的亲和力,能把脂肪酸从细胞膜转运到细胞内利用位点,在长链脂肪酸的代谢中起重要作用。本文就脂肪酸结合蛋白的结构、功能及其对脂肪酸代谢调节方面的研究进行了综述,并阐述了猪脂肪酸结合蛋白基因地对肌内脂肪合成的影响。     

Studies of the fatty acid-binding site of rat liver fatty acid-binding protein using fluorescent fatty acids

Rat liver fatty acid-binding protein (FABP) is a 14.3-kDa cytosolic protein which binds long chain free fatty acids (ffa) and is believed to participate in intracellular movement and/or distribution of ffa. In the studies described here fluorescently labeled ffa were used to examine the physical nature of the ffa-binding site on FABP. The fluorescent analogues were 16- and 18-carbon ffa with an anthracene moiety covalently attached at eight different points along the length of the hydrocarbon chain (AOffa). Emission maxima of all FABP-bound AOffa were found to be considerably blue-shifted with respect to emission of phospholipid membrane-bound AOffa, suggesting a high degree of motional constraint for protein-bound ffa. Large fluorescence quantum yields and long excited state life-times indicate that the FABP-binding site for ffa is highly hydrophobic. Analysis of rotational correlation times for the FABP-bound AOffa suggest that the ffa are tightly bound to the protein. Variation of the quantum yield with attachment site suggests that the carboxylic acid group of the fatty acyl chain is located near the aqueous surface of the FABP. The rest of the ffa hydrocarbon chain is buried within the protein in a hydrophobic pocket and is particularly constrained at the midportion of the acyl chain.     

Purification of murine adipocyte lipid-binding protein. Characterization as a fatty acid- and retinoic acid-binding protein

An adipose-specific protein has been purified from murine 3T3-L1 adipocytes to greater than 98% homogeneity. A purification procedure was developed utilizing a combination of gel filtration, cation exchange chromatography, and covalent chromatography on activated-thiol Sepharose 4B. The protein exists as a single polypeptide with a molecular weight of about 15,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein contains 2 mol of reduced sulfhydryl groups per mol of protein and an amino terminus blocked to sequencing. Automated Edman degradation of trypsin and CNBr-derived peptides has verified that the purified protein is that predicted by the mRNA (Bernlohr, D. A., Angus, C. W., Lane, M. D., Bolanowski, M. A., and Kelly, T. J. Jr. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5468-5472). Based on sequence analysis, the 15-kDa adipocyte protein is considered to be a member of a family of tissue-specific, cytosolic lipid-binding proteins. Utilizing a liposome assay, the purified protein binds both oleic acid and retinoic acid saturably with approximately 1 mol of ligand bound per mol of protein. Dissociation constants determined from Scatchard analysis were 3 and 50 microM, respectively. This report represents the first demonstration of a member of this family of structurally related proteins that is capable of binding both fatty acid and retinoic acid. Hence, we propose the name adipocyte lipid-binding protein, or ALBP.     

Characterization and amino acid sequence of a fatty acid-binding protein from human heart.

The complete amino acid sequence of a fatty acid-binding protein from human heart was determined by automated Edman degradation of CNBr, BNPS-skatole [3'-bromo-3-methyl-2-(2-nitrobenzenesulphenyl)indolenine], hydroxylamine, Staphylococcus aureus V8 proteinase, tryptic and chymotryptic peptides, and by digestion of the protein with carboxypeptidase A. The sequence of the blocked N-terminal tryptic peptide from citraconylated protein was determined by collisionally induced decomposition mass spectrometry. The protein contains 132 amino acid residues, is enriched with respect to threonine and lysine, lacks cysteine, has an acetylated valine residue at the N-terminus, and has an Mr of 14768 and an isoelectric point of 5.25. This protein contains two short internal repeated sequences from residues 48-54 and from residues 114-119 located within regions of predicted beta-structure and decreasing hydrophobicity. These short repeats are contained within two longer repeated regions from residues 48-60 and residues 114-125, which display 62% sequence similarity. These regions could accommodate the charged and uncharged moieties of long-chain fatty acids and may represent fatty acid-binding domains consistent with the finding that human heart fatty acid-binding protein binds 2 mol of oleate or palmitate/mol of protein. Detailed evidence for the amino acid sequences of the peptides has been deposited as Supplementary Publication SUP 50143 (23 pages) at the British Library Lending Division, Boston Spa, Yorkshire LS23 7BQ, U.K., from whom copies may be obtained as indicated in Biochem. J. (1988) 249, 5.     

ERK8, a new member of the mitogen-activated protein kinase family

The ERKs are a subfamily of the MAPKs that have been implicated in cell growth and differentiation. By using the rat ERK7 cDNA to screen a human multiple tissue cDNA library, we identified a new member of the ERK family, ERK8, that shares 69% amino acid sequence identity with ERK7. Northern analysis demonstrates that ERK8 is present in a number of tissues with maximal expression in the lung and kidney. Fluorescence in situ hybridization localized the ERK8 gene to chromosome 8, band q24.3. Expression of ERK8 in COS cells and bacteria indicates that, in contrast to constitutively active ERK7, ERK8 has minimal basal kinase activity and a unique substrate profile. ERK8, which contains two SH3-binding motifs in its C-terminal region, associates with the c-Src SH3 domain in vitro and co-immunoprecipitates with c-Src in vivo. Co-transfection with either v-Src or a constitutively active c-Src increases ERK8 activation indicating that ERK8 can be activated downstream of c-Src. ERK8 is also activated following serum stimulation, and the extent of this activation is reduced by pretreatment with the specific Src family inhibitor PP2. The ERK8 activation by serum or Src was not affected by the MEK inhibitor U0126 indicating that activation of ERK8 does not require MEK1, MEK2, or MEK5. Although most closely related to ERK7, the relatively low sequence identity, minimal basal activity, and different substrate profile identify ERK8 as a distinct member of the MAPK family that is activated by an Src-dependent signaling pathway.     

Calexcitin B is a new member of the sarcoplasmic calcium-binding protein family

Calexcitin (CE) is a calcium sensor protein that has been implicated in associative learning. The CE gene was previously cloned from the long-finned squid, Loligo pealei, and the gene product was shown to bind GTP and modulate K(+) channels and ryanodine receptors in a Ca(2+)-dependent manner. We cloned a new gene from L. pealei, which encodes a CE-like protein, here named calexcitin B (CE(B)). CE(B) has 95% amino acid identity to the original form. Our sequence analyses indicate that CEs are homologous to the sarcoplasmic calcium-binding protein subfamily of the EF-hand superfamily. Far and near UV circular dichroism and nuclear magnetic resonance studies demonstrate that CE(B) binds Ca(2+) and undergoes a conformational change. CE(B) is phosphorylated by protein kinase C, but not by casein kinase II. CE(B) does not bind GTP. Western blot experiments using polyclonal antibodies generated against CE(B) showed that CE(B) is expressed in the L. pealei optic lobe. Taken together, the neuronal protein CE represents the first example of a Ca(2+) sensor in the sarcoplasmic calcium-binding protein family.