The liver fatty acid binding protein - comparison of cavity properties of intracellular lipid-binding proteins

The crystal and solution structures of all of the intracellular lipid binding proteins (iLBPs) reveal a common -barrel framework with only small local perturbations. All existing evidence points to the binding cavity and a poorly delimited portal region as defining the function of each family member. The importance of local structure within the cavity appears to be its influence on binding affinity and specificity for the lipid. The portal region appears to be involved in the regulation of ligand exchange. Within the iLBP family, liver fatty acid binding protein or LFABP, has the unique property of binding two fatty acids within its internalized binding cavity rather than the commonly observed stoichiometry of one. Furthermore, LFABP will bind hydrophobic molecules larger than the ligands which will associate with other iLBPs. The crystal structure of LFABP contains two bound oleate molecules and provides the explanation for its unusual stoichiometry. One of the bound fatty acids is completely internalized and has its carboxylate interacting with an arginine and two serines. The second oleate represents an entirely new binding mode with the carboxylate on the surface of LFABP. The two oleates also interact with each other. Because of this interaction and its inner location, it appears the first oleate must be present before the second more external molecule is bound.     

Distinct Unfolding and Refolding Pathways of Lipid Transfer Proteins LTP1 and LTP2

Plant non-specific lipid transfer proteins (ns-LTPs) comprise two families, LTP1s and LTP2s, all structurally stabilized by four native disulfide bonds. Solution and crystal structures of both LTP1s and LTP2s from various plants have been determined. Despite the similarities of their biological function and backbone folds, the biophysical properties of LTP1s and LTP2s differ significantly. In this report, the mechanisms of unfolding and refolding of rice LTP1 and LTP2 have been investigated using the technique of disulfide bonds scrambling. LTP1 is shown to unfold and refold via predominant species of partially structured intermediates. Four isomers of partly unfolded and extensively unfolded LTP1 were identified, isolated and their disulfide structures were determined. By contrast, unfolding and refolding of LTP2 adopt a (close to) two-state mechanism, and undergo a reversible conversion between the native and a single extensively unfolded isomer without accumulation of any significant intermediate.     

Does Intrinsic Disorder in Proteins Favor Their Interaction with Lipids?

Intrinsically disordered proteins (IDPs) are implicated in a range of human diseases, some of which are associated with the ability to bind to lipids. Although the presence of solvent‐exposed hydrophobic regions in IDPs should favor their interactions with low‐molecular‐weight hydrophobic/amphiphilic compounds, this hypothesis has not been systematically explored as of yet. In this study, the analysis of the DisProt database with regard to the presence of lipid‐binding IDPs (LBIDPs) reveals that they comprise, at least, 15% of DisProt entries. LBIDPs are classified into four groups by ligand type, functional categories, domain structure, and conformational state. 57% of LBIDPs are classified as ordered according to the CH‐CDF analysis, and 70% of LBIDPs possess lengths of disordered regions below 50%. To investigate the lipid‐binding properties of IDPs for which lipid binding is not reported, three proteins from different conformational groups are rationally selected. They all are shown to bind linoleic (LA) and oleic (OA) acids with capacities ranging from 9 to 34 LA/OA molecules per protein molecule. The association with LA/OA causes the formation of high‐molecular‐weight lipid–protein complexes. These findings suggest that lipid binding is common among IDPs, which can favor their involvement in lipid metabolism.     

Fluorescent dyes as probes to study lipid-binding proteins

We studied the equilibrium binding of two hydrophobic fluorescent dyes, ANS and bisANS, to four members of a family of intracellular lipid-binding proteins: IFABP, CRABP I, CRABP II, and ILBP. The spectral and binding parameters for the probes bound to the proteins were determined. Typically, there was a single binding site on each protein for the ligands. However, IFABP cooperatively bound a second bisANS molecule in the binding pocket. Comparative analysis of affinities and spectral characteristics for the two probes allowed us to examine the contributions of electrostatic and hydrophobic interactions to the binding process, and to address some aspects of the internal structure of the studied proteins.     

膜上tRNA结合蛋白的分离与初步鉴定

用ＴｒｉｔｏｎＸ－１１４分相法分离啤酒酵母的膜总蛋白,经过酵母ｔＲＮＡ分子交联的Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析,用０－０．８ｍｏｌ／Ｌ（ＮＨ４０２ＳＯ４梯度缓冲液洗脱ｔＲＮＡ结合的蛋白质。凝胶阻滞电泳实验室鉴定出两种主要的与ｔＲＮＡ分子特异性结合的蛋白质。     

Detecting distant relatives of mammalian LPS-binding and lipid transport proteins.

In mammals, a family of four lipid binding proteins has been previously defined that includes two lipopolysaccharide binding proteins and two lipid transfer proteins. The first member of this family to have its three-dimensional structure determined is bactericidal/permeability-increasing protein (BPI). Using both the sequence and structure of BPI, along with recently developed sequence-sequence and sequence-structure similarity search methods, we have identified 13 distant members of the family in a diverse set of eukaryotes, including rat, chicken, Caenorhabditis elegans, and Biomphalaria galbrata. Although the sequence similarity between these 13 new members and any of the 4 original members of the BPI family is well below the "twilight zone," their high sequence-structure compatibility with BPI indicates they are likely to share its fold. These findings broaden the BPI family to include a member found in retina and brain, and suggest that a primitive member may have contained only one of the two similar domains of BPI.     

水稻非特异性脂质转移蛋白的原核表达、纯化及抑菌功能

将编码水稻非特异性脂质转移蛋白 (nonspecificlipidtransferprotein ,nsLTP)基因 (LTP110 )的克隆到硫氧还蛋白融合表达载体PET32a( )中 ,在BL2 1(DE3)trxB-宿主菌中实现了融合蛋白的高表达。通过Ni2 chelatingSepharosefastflow柱纯化融合蛋白后 ,通过肠激酶酶切再过该亲和柱得到了重组LTP110。CD谱扫描表明重组蛋白质与体内提取的nsLTP二级结构相似 ;荧光脂质结合实验表明该蛋白质具有结合脂肪酸分子的活性。对该蛋白质的抑菌功能进行研究后表明 ,LTP110具有抑制稻瘟病菌孢子萌发的功能 ,在较低浓度即能发挥活性     

钙调素对钙调素结合蛋白-10和玉米非特异性脂转移蛋白与脂质结合活性的影响

荧光标记的脂质结合实验表明,钙调素结合蛋白-10（CaMBP-10）具有典型的植物非特异性脂质转移蛋白与脂质结合的特性。进一步实验研究了钙调素（calmodulin,CaM）对CaMBP-10和玉米nsLTP与脂质结合的活性的影响,结果显示无论在有钙和无钙条件下,CaM对两者的影响均有不同之处,W-7和TFP能消除CaM的影响。提示CaM不仅与CaMBP-10和玉米nsLTP特异性相互作用,而且对2种脂转移蛋白可能具有不同的调节机制。     

Scavenging of superoxide anion radical by chaparral

Plant cells contain lipid-transfer proteins (LTPs) able to transfer phospholipids between membranes in vitro. Plant LTPs share in common structural and functional features. Recent structural studies carried out by NMR and X-ray crystallography on an LTP isolated from maize seeds have showed that this protein involves four helices packed against a C-terminal region and stabilized by four disulfide bridges. A most striking feature of this structure is the existence of an internal hydrophobic cavity running through the whole molecule and able to accomodate acyl chains. It was thus of interest to study the ability of maize LTP to bind hydrophobic ligands such as acyl chains or lysophosphatidylcholine and to determine the effect of this binding on phospholipid transfer. The binding abilities of maize LTP, presented in this paper, are discussed and compared to those of lipid-binding proteins from animal tissues.     

Chronic high-fat diet affects intestinal fat absorption and postprandial triglyceride levels in the mouse

The effects of chronic fat overconsumption on intestinal physiology and lipid metabolism remain elusive. It is unknown whether a fat-mediated adaptation to lipid absorption takes place. To address this issue, mice fed a high-fat diet (40%, w/w) were refed or not a control diet (3%, w/w) for 3 additive weeks. Despite daily lipid intake 7.7-fold higher than in controls, fecal lipid output remained unchanged in mice fed the triglyceride (TG)-rich diet. In situ isolated jejunal loops revealed greater [1-(14)C]linoleic acid uptake without TG accumulation in mucosa, suggesting an increase in lipid absorption capacity. Induction both in intestinal mitotic index and in the expression of genes involved in fatty acid uptake, trafficking, and lipoprotein synthesis was found in high-fat diet mice. These changes were lipid-mediated, in that they were fully abolished in mice refed the control diet. A lipid load test performed in the presence or absence of the LPL inhibitor tyloxapol showed a sustained blood TG clearance in fat-fed mice likely attributable to intestinal modulation of LPL regulators (apolipoproteins C-II and C-III). These data demonstrate that a chronic high-fat diet greatly affects intestinal physiology and body lipid use in the mouse.     

Crystal structure of peach Pru p 3, the prototypic member of the family of plant non-specific lipid transfer protein pan-allergens

This study describes the three-dimensional crystal structure of a non-specific lipid transport protein (ns-LTP) from Rosaceae. Whilst ns-LTPs from species other than Rosaceae, such as nuts, cereals, grape, oranges and vegetables are also responsible for plant food allergies, this is less frequent compared with ns-LTPs from Rosaceae in the Mediterranean area. In this heterologously expressed peach Pru p3, a ligand is present inside the central cavity of the protein, presumably a fatty acid that was present or produced in the culture medium of the expression organism Escherichia coli. Moreover, the two molecules of ns-LTP present in the asymmetric unit bind this ligand in a different way, suggesting a significant degree of plasticity for the peach ns-LTP binding cavity, despite the presence of four disulphide bridges. Two molecules are present in the asymmetric unit: molecule A is a fully liganded protein, while molecule B apparently represents a partially liganded state. Also, molecular dynamics simulation, along with other evidence, suggests that these two molecular conformations represent different states in solution. Comparison of the 3D models of different ns-LTPs justifies the evidence of a high degree of conservation of the putative IgE binding epitopes among proteins of the Rosaceae family and the presence of significant amino acid replacements in correspondence of the same regions in ns-LTPs of botanical species unrelated to Rosaceae.     

Nomenclature of fatty acid-binding proteins

Summary A variety of designations is currently being used to refer to cellular fatty acid-binding proteins (FABPs). Besides from the use of other general names (e.g. Z protein), confusion mostly arises from the application of various abbreviations and symbols to denote the tissue(s) of origin and cellular localization (cytoplasm, plasma membrane) of a specific FABP. In order to minimize confusion a more unified and rational nomenclature is proposed, which is based on application of the formula X-FABPy. The prefix X is a capital letter indicating the tissue of greatest abundance, the suffix Y similarly denotes the (sub)cellular localization of the protein. The general and functional name fatty acid-binding protein (FABP) is preferred for the cellular proteins with the property to bind fatty acids, unless future research reveals that the binding of fatty acids is not the primary biological property or physiological role of (some of) these proteins.     

The biochemistry and biology of extracellular plant lipid-transfer proteins (LTPs)

Plant lipid-transfer proteins (LTPs) are abundant, small, lipid binding proteins that are capable of exchanging lipids between membranes in vitro. Despite their name, a role in intracellular lipid transport is considered unlikely, based on their extracellular localization. A number of other biological roles, including antimicrobial defense, signaling, and cell wall loosening, have been proposed, but conclusive evidence is generally lacking, and these functions are not well correlated with in vitro activity or structure. A survey of sequenced plant genomes suggests that the two biochemically characterized families of LTPs are phylogenetically restricted to seed plants and are present as substantial gene families. This review aims to summarize the current understanding of LTP biochemistry, as well as the evidence supporting the proposed in vivo roles of these proteins within the emerging post-genomic framework.     

A Lipid Transfer Protein Signaling Axis Exerts Dual Control of Cell-Cycle and Membrane Trafficking Systems

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Visualization of phosphatidylinositol 4,5-bisphosphate in the plasma membrane of suspension-cultured tobacco BY-2 cells and whole Arabidopsis seedlings

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] is an important signalling lipid in mammalian cells, where it functions as a second-messenger precursor in response to agonist-dependent activation of phospholipase C (PLC) but also operates as a signalling molecule on its own. Much of the recent knowledge about it has come from a new technique to visualize PtdIns(4,5)P(2)in vivo, by expressing a green or yellow fluorescent protein (GFP or YFP) fused to the pleckstrin homology (PH) domain of human PLCdelta1 that specifically binds PtdIns(4,5)P(2). In this way, YFP-PH(PLCdelta1) has been shown to predominantly label the plasma membrane and to transiently translocate into the cytoplasm upon PLC activation in a variety of mammalian cell systems. In plants, biochemical studies have shown that PtdIns(4,5)P(2) is present in very small quantities, but knowledge of its localization and function is still very limited. In this study, we have used YFP-PH(PLCdelta1) to try monitoring PtdIns(4,5)P(2)/PLC signalling in stably-transformed tobacco Bright Yellow-2 (BY-2) cells and Arabidopsis seedlings. In both plant systems, no detrimental effects were observed, indicating that overexpression of the biosensor did not interfere with the function of PtdIns(4,5)P(2). Confocal imaging revealed that most of the YFP-PH(PLCdelta1) fluorescence was present in the cytoplasm, and not in the plasma membrane as in mammalian cells. Nonetheless, four conditions were found in which YFP-PH(PLCdelta1) was concentrated at the plasma membrane: (i) upon treatment with the PLC inhibitor U73122; (ii) in response to salt stress; (iii) as a gradient at the tip of growing root hairs; (iv) during the final stage of a BY-2 cell division. We conclude that PtdIns(4,5)P(2), as in animals, is present in the plasma membrane of plants, but that its concentration in most cells is too low to be detected by YFP-PH(PLCdelta1). Hence, the reporter remains unbound in the cytosol, making it unsuitable to monitor PLC signalling. Nonetheless, YFP-PH(PLCdelta1) is a valuable plant PtdIns(4,5)P(2) reporter, for it highlights specific cells and conditions where this lipid becomes abnormally concentrated in membranes, raising the question of what it is doing there. New roles for PtdIns(4,5)P(2) in plant cell signalling are discussed.     

昆虫信息素结合蛋白的研究概况

在昆虫感受信息素的嗅觉反应中,信息素结合蛋白发挥了重要的作用。它作为脂溶性信息素的溶剂和载体,在亲水性淋巴液中起着运载信息素和使之失活的双重作用。由于它在昆虫识别信息素物质中起着重要的作用,近1 0年来,国内外对其进行了广泛、深入的研究。文章从信息素结合蛋白的生化特点、表达情况、代谢以及生理功能等方面的概况进行综述。     

Prediction and validation of the unexplored RNA‐binding protein atlas of the human proteome

Detecting protein‐RNA interactions is challenging both experimentally and computationally because RNAs are large in number, diverse in cellular location and function, and flexible in structure. As a result, many RNA‐binding proteins (RBPs) remain to be identified. Here, a template‐based, function‐prediction technique SPOT‐Seq for RBPs is applied to human proteome and its result is validated by a recent proteomic experimental discovery of 860 mRNA‐binding proteins (mRBPs). The coverage (or sensitivity) is 42.6% for 1217 known RBPs annotated in the Gene Ontology and 43.6% for 860 newly discovered human mRBPs. Consistent sensitivity indicates the robust performance of SPOT‐Seq for predicting RBPs. More importantly, SPOT‐Seq detects 2418 novel RBPs in human proteome, 291 of which were validated by the newly discovered mRBP set. Among 291 validated novel RBPs, 61 are not homologous to any known RBPs. Successful validation of predicted novel RBPs permits us to further analysis of their phenotypic roles in disease pathways. The dataset of 2418 predicted novel RBPs along with confidence levels and complex structures is available at http://sparks-lab.org (in publications) for experimental confirmations and hypothesis generation. Proteins 2014; 82:640–647. © 2013 Wiley Periodicals, Inc.     

Insight into the stability of the hydrophobic binding proteins of Escherichia coli: assessing the proteins for use as biosensors

Spectroscopic methods were used to monitor the unfolding of the leucine specific (LS) and the leucine-isoleucine-valine (LIV) binding proteins. Our studies indicate that ligand-free protein undergoes a simple two-state unfolding, whereas the protein-ligand complex undergoes a three-state unfolding model. Ligand binding causes significant stabilization of both proteins. There is correlation between ligand hydrophobicity and protein stabilization: the most hydrophobic ligand, isoleucine, causes the most significant stabilization of LIV protein. A disulfide bond present in N-domain of both proteins makes a large contribution to the protein stability of these periplasmic binding receptors.     

Structure and function of cytoplasmic retinoid binding proteins

We examined the ligand protein interactions of two highly homologous cellular retinol binding proteins, CRBP and CRBP-II, and two highly homologous cellular retinoic acid binding proteins, CRABP-I and CRABP-II. While the crystal structures of all four have been determined, nuclear magnetic resonance studies provide a means for observing dynamic aspects of ligand protein interactions of these proteins in solution. The cellular functions of these proteins are less well understood. We have modeled retinoid flux between cytoplasmic retinoid proteins and model membranes and with nuclear receptors. Based on our in vitro studies, we propose that certain retinoids may indirectly influence retinoid signaling by displacing endogenous retinoids from the cytoplasmic proteins to the nuclear receptors.     

Novel classes of fatty acid and retinol binding protein from nematodes

Parasitic nematodes have recently been found to produce proteins which represent two new classes of fatty acid and retinoid binding protein. The first is the nematode polyprotein allergens/antigens (NPAs) which, as their name suggests, are synthesised as large polyproteins which are subsequently cleaved at regularly spaced sites to form multiple copies of a fatty acid binding protein of approximately 14.5 kDa. Binding studies using molecular environment-sensitive fluorescent ligands have shown that the binding site is highly unusual, producing blue-shifting in fluorescence to an unprecedented degree, suggesting a remarkably non-polar environment and isolation from solvent water. Computer-based structural predictions and biophysical observations have identified the NPAs as highly helical proteins which might form a four helix bundle, so constitute a new class of lipid binding protein from animals. The second class, like the NPAs, binds both fatty acids and retinol, but with a higher affinity for the latter. These are also highly helical but are structurally distinct from the NPAs. The biological function of these new classes of protein are discussed in the context of both the metabolic requirements of the parasites and the possible role of the proteins in control of the immune and inflammatory environment of the tissue sites parasitised.