Biochemical characterization of the interaction between HspA1A and phospholipids

Seventy-kilodalton heat shock proteins (Hsp70s) are molecular chaperones essential for maintaining cellular homeostasis. Apart from their indispensable roles in protein homeostasis, specific Hsp70s localize at the plasma membrane and bind to specific lipids. The interaction of Hsp70s with lipids has direct physiological outcomes including lysosomal rescue, microautophagy, and promotion of cell apoptosis. Despite these essential functions, the Hsp70-lipid interactions remain largely uncharacterized. In this study, we characterized the interaction of HspA1A, an inducible Hsp70, with five phospholipids. We first used high concentrations of potassium and established that HspA1A embeds in membranes when bound to all anionic lipids tested. Furthermore, we found that protein insertion is enhanced by increasing the saturation level of the lipids. Next, we determined that the nucleotide-binding domain (NBD) of the protein binds to lipids quantitatively more than the substrate-binding domain (SBD). However, for all lipids tested, the full-length protein is necessary for embedding. We also used calcium and reaction buffers equilibrated at different pH values and determined that electrostatic interactions alone may not fully explain the association of HspA1A with lipids. We then determined that lipid binding is inhibited by nucleotide-binding, but it is unaffected by protein-substrate binding. These results suggest that the HspA1A lipid-association is specific, depends on the physicochemical properties of the lipid, and is mediated by multiple molecular forces. These mechanistic details of the Hsp70-lipid interactions establish a framework of possible physiological functions as they relate to chaperone regulation and localization.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-015-0636-6) contains supplementary material, which is available to authorized users.     

Antibodies induced by liposomal protein exhibit dual binding to protein and lipid epitopes

Natural polyreactive antibodies can accommodate chemically unrelated epitopes, such as lipids and proteins, in a single antigen binding site. Because liposomes containing lipid A as an adjuvant can induce antibodies directed against specific lipids, we immunized mice with liposomes containing lipid A together with a protein or peptide antigen to determine whether monoclonal antibodies generated after immunization would be specifically directed both to the liposomal lipid (either cholesterol or galactosylceramide) and also to the accompanying liposomal protein or peptide. Monoclonal antibodies were obtained that bound, by ELISA, to cholesterol and to recombinant gp140 envelope protein from HIV-1, or to galactosylceramide and to an HIV-1 envelope peptide. Surface plasmon resonance studies with the former antibody showed that the liposomal cholesterol and liposomal gp140 each contributed to the overall binding energy of the antibody to liposomes containing cholesterol and protein.     

Isolation and characterization of novel presenilin binding protein

Approximately 50% of familial Alzheimer's disease (AD) cases are linked to the presenilin (PS) gene. This suggests that an altered function of mutated PSs accounts for a fundamental process leading to AD. Here we identify a new PS binding protein, PBP, which is highly expressed in cerebral cortex and hippocampus. immunohistochemical studies and cell fractionation analysis show that PBP redistributes from cytoplasm to membranes in the presence of PS. In addition, PBP is deficient in the soluble fraction of sporadic AD brains.     

中华蜜蜂气味结合蛋白AcerOBP14的表达及配体结合特性分析

[目的]气味结合蛋白(odorant binding proteins,OBPs)在昆虫寄主定位、产卵地选择等行为中发挥着重要作用,明确中华蜜蜂Apis cerana cerana AcerOBP14与配体的结合特性有助于阐明中华蜜蜂嗅觉识别的分子机制.[方法]通过qRT-PCR测定OBP14在20日龄中华蜜蜂成年工蜂...     

Metal complexes of naphthoquinone based ligand: synthesis,characterization, protein binding,DNA binding/cleavage and cytotoxicity studies

Protein binding, DNA binding/cleavage and in vitro cytotoxicity studies of 2-((3-(dimethylamino)propyl)amino)naphthalene-1,4-dione (L) and its four coordinated M(II) complexes [M(II) = Co(II), Cu(II), Ni(II) and Zn(II)] have been investigated using various spectral techniques. The structure of the ligand was confirmed by spectral and single crystal XRD studies. The geometry of the complexes has been established using analytical and spectral investigations. These complexes show good binding tendency to bovine serum albumin (BSA) exhibiting high binding constant values (10⁵ M^?1) when compared to free ligand. Fluorescence titration studies reveal that these compounds bind strongly with CT-DNA through intercalative mode (K_app 10⁵ M^?1) and follow the order: Cu(II) > Zn(II) > Ni(II) > Co(II) > L. Molecular docking study substantiate the strength and mode of binding of these compounds with DNA. All the complexes efficiently cleaved pUC18-DNA via hydroxyl radical mechanism and the Cu(II) complex degraded the DNA completely by converting supercoiled form to linear form. The complexes demonstrate a comparable in vitro cytotoxic activity against two human cancer cell lines (MCF-7 and A-549), which is comparable with that of cisplatin. AO/EB and DAPI staining studies suggest apoptotic mode of cell death, in these cancer cells, with the compounds under investigation.     

乙肝表面抗原结合蛋白SBP在毕赤酵母中的表达纯化及功能鉴定

乙肝表面抗原结合蛋白(HBsAg binding protein,SBP)是本实验室发现的一种人源蛋白,该蛋白与人乙型肝炎病毒HBV表面抗原HBsAg存在特异性的结合能力。此前的研究证实SBP具有增强乙肝疫苗免疫效果的作用。为进一步研究该蛋白的生理功能和作用机制,利用毕赤酵母表达系统进行了SBP的表达菌株构建,筛选得到了SBP的高效表达菌株。发酵产物经过分离纯化,最终得到了大量高纯度的真核来源的目的蛋白。通过SDS-PAGE、高效液相色谱、Western blotting和质谱鉴定,证实所得到的蛋白具有较高的纯度和完整性。通过ELISA方法初步证实了其与乙肝表面抗原具有较好的结合能力。该研究为进一步进行SBP的体内外功能研究及免疫增效研究打下了基础。     

Automated on-like dialysis, trace enrichment and high-performance liquid chromatography Inhibition of interaction with the dialysis membrane and disruption of protein binding in the determination of clozapine in human plasma

Problems related to interaction of drugs with the dialysis membrane and to protein binding must be overcome in order to develop automated methods for drug analysis based on on-line dialysis, trace enrichment and HPLC. In order to study these problems, clozapine and its active metabolite N-desmethylclozapine were chosen as model compounds because they were found to interact with the dialysis membrane, and clozapine is highly protein bound. Addition of a cationic surfactant, dodecylethyldimethyl ammonium bromide, to the donor solution and to the plasma samples was found to inhibit interaction of the drugs with surfaces. The protein binding in plasma was disrupted prior to dialysis by lowering the pH with hydrochloric acid and the plasma proteins were solubilised with glycerol. The results obtained were used to develop a fully automated method for the determination of clozapine and N-desmethylclozapine in human plasma. More than 100 samples could be analysed within 24 h. The limit of detection in human plasma was 0.050 μmol/1 for clozapine and 0.055 μmol/1 for N-desmethylclozapine. Linearity was found for drug concentrations between 0.25–3 μmol/1. The relative standard deviations were between 1.2–6.7% and the method was applicable for therapeutic drug monitoring.     

Molecular characterization of protein kinase C-alpha binding to lamin A

Previous results from our laboratory have identified lamin A as a protein kinase C (PKC)-binding protein. Here, we have identified the regions of PKC-alpha that are crucial for this binding. By means of overlay assays and fusion proteins made of glutathione-S-transferase (GST) fused to elements of rat PKC-alpha, we have established that binding occurs through both the V5 region and a portion of the C2 region (i.e., the calcium-dependent lipid binding (CaLB) domain) of the kinase. In particular, we have found that amino acid 200-217 of the CaLB domain are essential for binding lamin A, as a synthetic peptide corresponding to this stretch of amino acids prevented the interaction between the CaLB domain and lamin A. We also show that the presence of four lysine residues of the CaLB domain (K205, K209, K211, and K213) was essential for the binding. We have determined that binding of elements of PKC-alpha to lamin A does not require the presence of cofactors such as phosphatidylserine (PS) and Ca(2+). We have also found that the binding site of lamin A for the CaLB domain of PKC-alpha is localized in the carboxyl-terminus of the lamin, downstream of amino acid 499. Our findings may prove to be important to clarify the mechanisms regulating PKC function within the nucleus and may also lead to the synthesis of isozyme-specific drugs to attenuate or reverse PKC-dependent nuclear signaling pathways important for the pathogenesis of cancer.     

Carnitine palmitoyltransferase activities: Effects of serum albumin,acyl-CoA binding protein and fatty acid binding protein

The carnitine palmitoyltransferase activity of various subcellular preparations measured with octanoyl-CoA as substrate was markedly increased by bovine serum albumin at low M concentrations of octanoyl-CoA. However, even a large excess (500 M) of this acyl-CoA did not inhibit the activity of the mitochondrial outer carnitine palmitoyltransferase, a carnitine palmitoyltransferase isoform that is particularly sensitive to inhibition by low M concentrations of palmitoyl-CoA. This bovine serum albumin stimulation was independent of the salt activation of the carnitine palmitoyltransferase activity. The effects of acyl-CoA binding protein (ACBP) and the fatty acid binding protein were also examined with palmitoyl-CoA as substrate. The results were in line with the findings of stronger binding of acyl-CoA to ACBP but showed that fatty acid binding protein also binds acyl-CoA esters. Although the effects of these proteins on the outer mitochondrial carnitine palmitoyltransferase activity and its malonyl-CoA inhibition varied with the experimental conditions, they showed that the various carnitine palmitoyltransferase preparations are effectively able to use palmitoyl-CoA bound to ACBP in a near physiological molar ratio of 1:1 as well as that bound to the fatty acid binding protein. It is suggested that the three proteins mentioned above effect the carnitine palmitoyltransferase activities not only by binding of acyl-CoAs, preventing acyl-CoA inhibition, but also by facilitating the removal of the acylcarnitine product from carnitine palmitoyltransferase. These results support the possibility that the acyl-CoA binding ability of acyl-CoA binding protein and of fatty acid binding protein have a role in acyl-CoA metabolismin vivo.Abbreviations ACBP acyl-CoA binding protein - BSA bovine serum albumin - CPT carnitine palmitoyltransferase - CPT₀ malonyl-CoA sensitive CPT of the outer mitochondrial membrane - CPT malonyl-CoA insensitive CPT of the inner mitochondrial membrane - OG octylglucoside - OMV outer membrane vesicles - IMV inner membrane vesicles Affiliated to the Department of Experimental Medicine, University of Montreal     

Charge modification at conserved positively charged residues of fatty acid binding protein (FABP) from the giant liver fluke Fasciola gigantica: its effect on oligomerization and binding properties

Fatty acid binding proteins (FABPs) are capable of binding hydrophobic ligands with high affinity; thereby facilitating the cellular uptake and intracellular trafficking of fatty acids. In this study, functional characteristics of a cytoplasmic FABP from the giant liver fluke Fasciola gigantica (FgFABP) were determined. Binding of a fluorescent fatty acid analogue 11-[[5-dimethy aminonaphtalene-1-sulphonyl] amino] undecanoic acid (DAUDA) to FgFABP resulted in changes in the emission spectrum. The optimal excitation wavelength and maximum emission of fluorescence for binding activities with DAUDA were 350 nm and 550 nm, respectively. The binding activity for DAUDA was determined from titration experiments and revealed a K_d value of 2.95 ± 0.54 μM. Furthermore, we found that cross-linking profile of FgFABP with dithiobis-(succinimidylpropionate) (DSP) in the presence of DAUDA resulted in increased formation of higher-ordered oligomers compared to that in the absence of DAUDA. We also replaced five highly conserved positively charged residues (K9, K58, K91, R107 and K131) with alanine and studied their oligomerization and binding properties of the modified FgFABPs. The obtained data demonstrate that these residues do not appear to be involved in oligomerization. However, the K58A and R107A substitutions exhibited a reduction in binding affinities. K91A and R107A revealed an increase in maximal specific binding.     

家蚕保幼激素结合蛋白Bmtol基因的克隆及功能分析

家蚕头部是一个神经中枢和感受的器官,其头部含有触角和感觉毛,感受外界的信号,并将外界信号传送到大脑进行反应。保幼激素主要是由咽侧体合成和分泌的,而保幼激素结合蛋白是保幼激素转运和发挥功能的载体,在昆虫体内具有极其重要的功能。文中通过Silk DB和NCBI数据库筛选并鉴定到一个新的具有保幼激素结合蛋白家族保守结构的蛋白Bm TOL,其编码基因编号为BGIBMGA003404(Gen Bank登录号:KY681053)。利用原核表达系统成功表达了该蛋白,通过Ni-NTA亲和层析的方法获得了Bm TOL的重组蛋白并制备了多克隆抗体。组织表达分析发现无论是转录水平还是蛋白水平Bm TOL在头部都是高量表达,且Bmtol基因在起蚕时表达量较高,在5龄和蛹期表达量较低,而在化蛾后表达量又开始上调。免疫组化结果显示Bm TOL蛋白定位在头部的皮层、触角和脑中,推测其可能与头部信息传递有关,为家蚕的生长发育和行为调控提供重要的信息来源。     

Functional and receptor binding characterization of recombinant murine macrophage inflammatory protein 2: sequence analysis and mutagenesis identify receptor binding epitopes.

Murine macrophage inflammatory protein-2 (MIP-2), a member of the alpha-chemokine family, is one of several proteins secreted by cells in response to lipopolysaccharide. Many of the alpha-chemokines, such as interleukin-8, gro-alpha/MGSA, and neutrophil activating peptide-2 (NAP-2), are associated with neutrophil activation and chemotaxis. We describe the expression, purification, and characterization of murine MIP-2 from Pichia pastoris. Circular dichroism spectroscopy reveals that MIP-2 exhibits a highly ordered secondary structure consistent with the alpha/beta structures of other chemokines. Recombinant MIP-2 is chemotactic for human and murine neutrophils and up-regulates cell surface expression of Mac-1. MIP-2 binds to human and murine neutrophils with dissociation constants of 6.4 nM and 2.9 nM, respectively. We further characterize the binding of MIP-2 to the human types A and B IL-8 receptors and the murine homologue of the IL-8 receptor. MIP-2 displays low-affinity binding to the type A IL-8 receptor (Kd > 120 nM) and high-affinity binding to the type B IL-8 receptor (Kd 5.7 nM) and the murine receptor (Kd 6.8 nM). The three-dimensional structure of IL-8 and sequence analysis of six chemokines (IL-8, gro-alpha, NAP-2, ENA-78, KC, and MIP-2) that display high-affinity binding to the IL-8 type B receptor are used to identify an extended N-terminal surface that interacts with this receptor. Two mutants of MIP-2 establish that this region is also involved in binding and activating the murine homologue of the IL-8 receptor. Differences in the sequence between IL-8 and related chemokines identify a unique hydrophobic/aromatic region surrounded by charged residues that is likely to impart specificity to IL-8 for binding to the type A receptor.     

Molecular characterization,expression pattern and ligand‐binding properties of the pheromone‐binding protein gene from Cyrtotrachelus buqueti

Pheromone‐binding proteins (PBPs) play important roles in the information exchange between insect sexes, specifically in the process of transporting fat‐soluble odour molecules from the external environment to olfactory receptors through the olfactory sensillum lymph. The PBP functions in this process may explain the sex pheromone identification mechanism used by insects, laying a theoretical foundation for the prevention and control of pests by interfering with olfactory recognition. In the present study, a PBP gene of Cyrtotrachelus buqueti (GenBank accession number: KU845733) is cloned for prokaryotic expression. Using N‐phenyl‐1‐naphthylamine as the fluorescent probe in a competitive binding assay, the ability of CbuqPBP1 to bind 12 sex pheromone analogues and three volatiles of Neosinocalamus affinis shoots is examined. Of the 12 C. buqueti sex pheromone analogues, dibutyl phthalate gives the greatest displacement (inhibitory constant value of 11.1 μm ), whereas the other sex pheromone components show much smaller displacements. Consistent with other PBPs, the three plant volatiles (linalool, benzaldehyde and indole) show only a limited displacement of CbuqPBP1. However, the binding abilities of 1 : 1 ratios of each of the three plant volatiles with dibutyl phthalate show increases of 62.3%, 65.1% and 51.7% over the binding abilities of the three plant volatiles alone. CbuqPBP1 has dual roles in the processes of sensing sex pheromones and plant volatiles.     

家蚕化学感受蛋白CSP16的表达及结合特性分析

【目的】化学感受蛋白(chemosensory proteins,CSPs)在昆虫化学信号的感受识别和生长发育调节等生理过程具有重要作用。本研究旨在探索CSP16在家蚕Bombyx mori中的功能。【方法】利用RT-q PCR分析csp16在家蚕不同发育时期和不同组织的表达特征,用原核表达系统对家蚕CSP16进行表达纯化,并通过荧光结合实验检测该蛋白与不同配体化合物的结合特性。【结果】RT-q PCR结果表明,csp16在家蚕1-5龄幼虫中呈规律性表达,各龄期眠蚕中表达量最高,5龄第3天幼虫中主要表达于头、表皮、精巢和卵巢等。蜕皮激素(20E)处理后csp16在不同龄期幼虫和5龄幼虫不同组织中的表达量上调。纯化后CSP16的荧光结合实验表明,CSP16与醇类、酯类、醛类、酚类和苯环类等化合物的亲和力都较弱。【结论】csp16在家蚕不同龄期幼虫眠蚕中表达量最高,蜕皮激素使csp16在家蚕取食幼虫中的表达量出现上调,提示其可能参与家蚕幼虫的蜕皮过程。     

Molecular dynamics simulations of apo and holo forms of fatty acid binding protein 5 and cellular retinoic acid binding protein II reveal highly mobile protein,retinoic acid ligand,and water molecules

Structural and dynamic properties from a series of 300 ns molecular dynamics, MD, simulations of two intracellular lipid binding proteins, iLBPs, (Fatty Acid Binding Protein 5, FABP5, and Cellular Retinoic Acid Binding Protein II, CRABP-II) in both the apo form and when bound with retinoic acid reveal a high degree of protein and ligand flexibility. The ratio of FABP5 to CRABP-II in a cell may determine whether it undergoes natural apoptosis or unrestricted cell growth in the presence of retinoic acid. As a result, FABP5 is a promising target for cancer therapy. The MD simulations presented here reveal distinct differences in the two proteins and provide insight into the binding mechanism. CRABP-II is a much larger, more flexible protein that closes upon ligand binding, where FABP5 transitions to an open state in the holo form. The traditional understanding obtained from crystal structures of the gap between two β-sheets of the β-barrel common to iLBPs and the α-helix cap that forms the portal to the binding pocket is insufficient for describing protein conformation (open vs. closed) or ligand entry and exit. When the high degree of mobility between multiple conformations of both the ligand and protein are examined via MD simulation, a new mode of ligand motion that improves understanding of binding dynamics is revealed.     

Sequence-independent control of peptide conformation in liposomal vaccines for targeting protein misfolding diseases

Synthetic peptide immunogens that mimic the conformation of a target epitope of pathological relevance offer the possibility to precisely control the immune response specificity. Here, we performed conformational analyses using a panel of peptides in order to investigate the key parameters controlling their conformation upon integration into liposomal bilayers. These revealed that the peptide lipidation pattern, the lipid anchor chain length, and the liposome surface charge all significantly alter peptide conformation. Peptide aggregation could also be modulated post-liposome assembly by the addition of distinct small molecule β-sheet breakers. Immunization of both mice and monkeys with a model liposomal vaccine containing β-sheet aggregated lipopeptide (Palm1-15) induced polyclonal IgG antibodies that specifically recognized β-sheet multimers over monomer or non-pathological native protein. The rational design of liposome-bound peptide immunogens with defined conformation opens up the possibility to generate vaccines against a range of protein misfolding diseases, such as Alzheimer disease.     

多异瓢虫气味结合蛋白HvarOBP2的基因克隆及配体结合特性分析

【目的】本研究旨在克隆多异瓢虫Hippodamia variegata气味结合蛋白(odorant-binding proteins, OBPs)基因HvarOBP2,并解析这一蛋白的配体结合特征。【方法】采用BLAST和生物信息学技术从多异瓢虫成虫触角转录组中鉴定候选气味结合蛋白基因HvarOBP2,PCR特异扩增HvarOBP2基因全长cDNA序列;通过体外原核表达和亲和柱层析等手段获得HvarOBP2重组蛋白;运用荧光竞争结合实验测定重组蛋白HvarOBP2与40种植物挥发物组分以及24种蚜虫相关挥发物组分的结合能力;使用PyMOL1.9.0进行重组蛋白HvarOBP2与4种配体(β-紫罗兰酮、邻苯二甲酸二丁酯、油酸和橙花叔醇)的分子对接模拟。【结果】HvarOBP2(GenBank登录号：OK340816)开放阅读框(open reading frame, ORF)长447 bp,其编码蛋白N端有17个氨基酸组成的信号肽,具有6个保守的半胱氨酸位点,属于classical OBPs亚家族。荧光竞争结合实验结果表明,重组蛋白HvarOBP2与植物挥发物组分油酸(解离常数K