新型人血清白蛋白特异结合肽ML的筛选及鉴定

利用噬菌体展示技术筛选和鉴定新型人血清白蛋白(HSA)特异结合7肽,分析与垂体腺苷酸环化酶激活肽27(PACAP27)构成的融合多肽ML-PACAP27与HSA的亲和结合力,以确定筛选的7肽(ML1和ML2)在与其他药物多肽或蛋白融合状态下仍具有较高的HSA结合活力。利用噬菌体展示7肽库,经四轮筛选、筛选克隆的DNA测序、酶联免疫吸附技术分析(ELISA)初步鉴定筛选克隆与HSA的亲和作用,并利用等离子共振技术(SPR)定量测定合成的ML-PACAP27融合多肽与人血清白蛋白的亲和力常数。实验结果表明:经筛选获得2个与人血清白蛋白特异性结合的7肽序列,其氨基酸序列分别为:ML1:LKSCKPL和ML2:SLKSHAL;ELISA分析结果显示,含有ML1和ML2的噬菌体均可亲和结合HSA,而且ML2的亲和结合作用高于ML1;SPR分析结果显示,ML1-PACAP27和ML2-PACAP27与HSA的解离常数(KD)分别为:8.1×10-6mol/L和3.7×10-6mol/L,ML1-PACAP27与HSA的结合力高于ML1-PACAP27。筛选和鉴定了两个可与HSA特异性结合的7肽序列,该7肽序列可用于特异偶联HSA的长效分子药物的重组融合表达或设计,可为延长分子药物的半衰期及新型药物研发提供新工具。     

Identification and Characterization of Unique Proline-rich Peptides Binding to the Mitochondrial Fission Protein hFis1

Mammalian mitochondrial fission requires at least two proteins, hFis1 and the dynamin-like GTPase DLP1/Drp1. The mitochondrial protein hFis1 is anchored at the outer membrane by a C-terminal transmembrane domain. The cytosolic domain of hFis1 contains six α helices [α1–α6] out of which [α2–α5] form tetratricopeptide repeat (TPR)-like motifs. DLP1 and possibly other proteins are thought to interact with the hFis1 TPR region during the fission process. It has also been suggested that the α1-helix regulates protein-protein interactions at the TPR. We performed random peptide phage display screening using the hFis1[α2–α6] as the target and identified ten different peptide sequences. Phage ELISA using mutant hFis1 indicates that the peptide binding requires the α2 and α3 helices and the intact TPR structure. Competition experiments and surface plasmon resonance analyses confirmed that a subset of free peptides enriched with proline residues directly bind to the target. Two of these peptides bind to the α1-containing intact cytosolic domain of hFis1 with decreased affinity. Peptide microinjection into cells abolished the mitochondrial swelling induced by overexpression of α1-deleted hFis1, and significantly decreased cytochrome c release from mitochondria upon apoptotic induction. Our data demonstrate that hFis1 can bind to multiple amino acid sequences selectively, and that the TPR constitutes the main binding region of hFis1, providing a first insight into the hFis1 TPR as a potential therapeutic target.     

Screening and Identification of the Binding Peptides of Mycoplasma genitalium Protein of Adhesion

International Journal of Peptide Research and Therapeutics - Mycoplasma genitalium protein of adhesion (MgPa) is a vital membrane protein, which plays an important role in mediating the adhesion...     

人类蛋白质组表达谱蛋白质鉴定的分步搜索策略

大规模蛋白质组表达谱研究的蛋白质鉴定一般采取基于数据库搜索的策略,因此数据库的选择及搜索策略在蛋白质鉴定中非常重要。现有的人类蛋白质数据库远不够完善,而从其他物种的蛋白质数据库中所能得到的补充非常有限,但人类基因组数据库中却可能含有很大的补充空间。在对国际人类蛋白质数据库充分调研、比较的基础上,提出了一种分步搜索的策略。这种策略首先利用一个质量较高、覆盖率相对较大的非冗余数据库进行基本鉴定,随后利用其他蛋白和核酸数据库进行补充鉴定和新蛋白挖掘。该策略能有效地鉴定尽可能多的高可靠蛋白,并能进一步充分利用质谱数据进行补充鉴定和新蛋白挖掘,对大规模蛋白质组表达谱研究具有重要的意义。     

脑膜炎大肠杆菌IbeA蛋白结合肽序列的亲和筛选

应用噬菌体展示肽库技术,以重组的脑膜炎大肠杆菌致病蛋白IbeA作为靶分子,经过吸附-洗脱-扩增-再吸附的亲和筛选,随机挑选亲和力强的噬菌体克隆,进行ELISA、竞争抑制实验和序列测定。结果显示,经3轮淘选后,间接ELISA鉴定得到高亲和性结合IbeA蛋白的15个阳性克隆。竞争抑制实验结果表明,游离IbeA蛋白能竞争抑制噬菌体结合肽克隆与固相包被的IbeA蛋白的结合,其抑制作用随游离IbeA蛋白浓度的降低而减弱。测序结果得到5种阳性噬菌体克隆展示肽序列。上述结果提示以脑膜炎大肠杆菌IbeA蛋白为靶筛选所获得的噬菌体12肽克隆,具有特异性,其结合肽序列呈现相对保守性。建立的从噬菌体随机肽库筛选IbeA蛋白结合肽的方法具有方便、灵活和高效可行的特点。     

Binding of Human GM-CSF to Synthetic Peptides of the Alpha Subunit of Its Receptor

Abstract

We have synthesized a series of peptides corresponding to portions of the extracellular domain of human granulocyte-macrophage colony stimulating factor receptor α subunit (hGM-CSFRα). The sequences were chosen according to the homology between hGM-CSFRα and the growth hormone receptor (GHR) and correspond to the regions reported to form the binding site of the latter receptor. The peptides were examined for their binding activity to hGM-CSF by affinity chromatography on resin-immobilized hGM-CSF and by a solid phase binding assay. Four peptides endowed with hGM-CSF binding activity were identified and the postulated homology between the binding sites of hGM-CSFRα and GHR was confirmed.     

Whole-Cell Protein Identification Using the Concept of Unique Peptides

A concept of unique peptides(CUP)was proposed and implemented to identify whole-cell proteins from tandem mass spectrometry(MS/MS)ion spectra.A unique peptide is defined as a peptide,irrespective of its length,that exists only in one protein of a proteome of interest,despite the fact that this peptide may appear more than once in the same protein.Integrating CUP,a two-step whole-cell protein identification strategy was developed to further increase the confidence of identified proteins.A dataset containing 40,243 MS/MS ion spectra of Saccharomyces cerevisiae and protein identification tools including Mascot and SEQUEST were used to illustrate the proposed concept and strategy.Without implementing CUP,the proteins identified by SEQUEST are 2.26 fold of those identified by Mascot.When CUP was applied,the proteins bearing unique peptides identified by SEQUEST are3.89 fold of those identified by Mascot.By cross-comparing two sets of identified proteins,only 89 common proteins derived from CUP were found.The key discrepancy between identified proteins was resulted from the filtering criteria employed by each protein identification tool.According to the origin of peptides classified by CUP and the commonality of proteins recognized by protein identification tools,all identified proteins were cross-compared,resulting in four groups of proteins possessing different levels of assigned confidence.     

血管内皮细胞生长因子受体Flt-1结合小肽的融合表达及活性鉴定

血管内皮细胞生长因子 (VEGF)通过结合其酪氨酸激酶受体KDR、fms样酪氨酸激酶 1(Flt 1)调节新生血管形成 ;筛选能封闭VEGF结合Flt 1的小肽 ,可以通过阻断肿瘤血管形成 ,抑制实体瘤生长 .将从噬菌体 12肽库中筛选获得的 2个能与Flt 1结合的阳性噬菌体克隆 (F5 6和F90 )十二肽DNA(36bp)克隆到表达载体pQE4 2中 ,在大肠杆菌M15中稳定表达二氢叶酸还原酶融合蛋白(DHFR F5 6 F90 ) ,经变性、复性后得到纯度达 90 %的可溶性蛋白 .ELISA检测表明 ,DHFR F5 6 F90能结合可溶性受体sFlt 1和血管内皮细胞 ;12 5I VEGF竞争抑制实验显示 ,DHFR F5 6能竞争抑制VEGF同可溶性受体sFlt 1结合 .结果提示 ,F5 6可能是VEGF受体Flt 1的有效拮抗剂 ,具有抗肿瘤新生血管形成的潜在应用前景     

鉴定人甲基化CpG结合结构域蛋白4为蛋白激酶X的底物

人蛋白激酶X(PrKX)是由X染色体编码的一种cAMP依赖性蛋白激酶,但是到目前为止已鉴定到的PrKX底物还很少.为了鉴定蛋白激酶X的底物,我们以蛋白激酶X为诱饵进行了酵母双杂交实验,结果发现甲基化CpG结合结构域蛋白4(MBD4)与PrKX在酵母细胞内相互作用较强.GST融合蛋白沉降和细胞内蛋白质的免疫共沉淀证实PrKX与MBD4之间确实存在相互作用.进一步研究表明,大肠杆菌中表达的重组MBD4在体外可以被PrKX磷酸化,而且MBD4蛋白的磷酸化能明显增强它在体外与甲基化DNA探针的结合活性.     

Functional Expression of hMYH, a Human Homolog of the Escherichia coli MutY Protein

We have previously described the hMYH cDNA and genomic clones (M. M. Slupska et al., J. Bacteriol. 178:3885-3892, 1996). Here, we report that the enzyme expressed from an hMYH cDNA clone in Escherichia coli complements the mutator phenotype in a mutY mutant and can remove A from an A. 8-hydroxydeoxyguanine mismatch and to a lesser extent can remove A from an A. G mismatch in vitro.     

Predicting DNA-Binding Proteins and Binding Residues by Complex Structure Prediction and Application to Human Proteome

As more and more protein sequences are uncovered from increasingly inexpensive sequencing techniques, an urgent task is to find their functions. This work presents a highly reliable computational technique for predicting DNA-binding function at the level of protein-DNA complex structures, rather than low-resolution two-state prediction of DNA-binding as most existing techniques do. The method first predicts protein-DNA complex structure by utilizing the template-based structure prediction technique HHblits, followed by binding affinity prediction based on a knowledge-based energy function (Distance-scaled finite ideal-gas reference state for protein-DNA interactions). A leave-one-out cross validation of the method based on 179 DNA-binding and 3797 non-binding protein domains achieves a Matthews correlation coefficient (MCC) of 0.77 with high precision (94%) and high sensitivity (65%). We further found 51% sensitivity for 82 newly determined structures of DNA-binding proteins and 56% sensitivity for the human proteome. In addition, the method provides a reasonably accurate prediction of DNA-binding residues in proteins based on predicted DNA-binding complex structures. Its application to human proteome leads to more than 300 novel DNA-binding proteins; some of these predicted structures were validated by known structures of homologous proteins in APO forms. The method [SPOT-Seq (DNA)] is available as an on-line server at http://sparks-lab.org.     

Nucleotide Binding to the Human Multidrug Resistance Protein 3, MRP3

We used a spin-labeled ATP analog, SL-ATP, to study nucleotide binding to highly purified human multidrug resistance protein 3, MRP3, which had been expressed in the yeast Pichia pastoris. SL-ATP was shown to be a good substrate analog and is hydrolyzed by MRP3 at about 10% of the Vmax for normal ATP. ESR titrations showed that 2 mol of SL-ATP readily bound per mole of MRP3 with a dissociation constant of about 100 μM in the presence of Mg²⁺ ions. The binding curve was easily fitted for a hyperbolic binding relationship. SL-ATP also bound readily to MRP3 in the absence of divalent ions and presence of EDTA. The resulting binding curve, however, could not be satisfactorily fitted using the equation for hyperbola. Analysis showed that a good fit was only obtained with the Hill equation using a Hill coefficient of 4 or close to 4. Lower Hill coefficients resulted in lower goodness of the fit. Such cooperative binding may be explained by a dimerization event triggered in the absence of divalent ions and a close communication of nucleotide binding sites of the interacting dimers. These findings may be of great importance for the overall mechanism and regulation of multidrug resistance proteins.     

人DNAJB6蛋白与人巨细胞病毒皮层蛋白pUL23相互作用的鉴定

pUL23是人巨细胞病毒（HCMV）UL23基因编码的皮层蛋白. HCMV皮层蛋白与病毒颗粒的形成、病毒转移、免疫调控等病毒生活过程相关.利用GAL4 酵母双杂交系统筛选人胚肾cDNA文库,获得与人巨细胞病毒皮层蛋白pUL23相互作用的宿主蛋白分子DNAJB6 ［DnaJ (Hsp40) homolog, subfamily B, member 6］.回复酵母双杂交、体外GST-Pull down和免疫共沉淀试验再次确认两者之间的相互作用.该结果为进一步研究pUL23蛋白在HCMV生活周期中的作用机制提供依据.     

人胚胎干细胞膜的特异多肽受体研究

目的:通过多肽筛选和比较分析,找到针对人胚胎干细胞(hESC)特异结合的多肽的膜受体蛋白,为相关通路或特异膜表面蛋白下游的研究奠定基础。方法:首先,在前期运用噬菌体展示技术的基础上进行ELISA重筛选,通过对比结合强度的大小,挑选出特异性结合人胚胎干细胞的噬菌体多肽并且进行测序和合成有poly-his标签的多肽;然后运用His Pull-Down系统获得特异结合人胚胎干细胞的某一特殊噬菌体12肽的膜上靶分子受体蛋白;最后质谱测序后通过Mascot数据库和NCBI进行序列信息分析。结果:1通过ELISA重筛选,得到了高特异性结合人胚胎干细胞的两个噬菌体序列,其序列分别为HGAAWGTRTGHV(HGA)和VPATETAQAGHA(VPA)。2通过His Pull-Down实验得到了一个针对多肽VPA的特异性膜蛋白受体。3通过MALD质谱分析以及NCBI的数据库搜索分析,进一步确认这一VPA多肽特异性结合的潜在受体蛋白可能属于HECT超级家族。结论:寻找到一个潜在未知的人胚胎干细胞的特异表面标志物,此膜受体可与VPA多肽特异性结合,为人胚胎干细胞的筛选和鉴定提供了重要指标。     

Structural Basis for Parathyroid Hormone-related Protein Binding to the Parathyroid Hormone Receptor and Design of Conformation-selective Peptides

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are two related peptides that control calcium/phosphate homeostasis and bone development, respectively, through activation of the PTH/PTHrP receptor (PTH1R), a class B G protein-coupled receptor. Both peptides hold clinical interest for their capacities to stimulate bone formation. PTH and PTHrP display different selectivity for two distinct PTH1R conformations, but how their binding to the receptor differs is unclear. The high resolution crystal structure of PTHrP bound to the extracellular domain (ECD) of PTH1R reveals that PTHrP binds as an amphipathic α-helix to the same hydrophobic groove in the ECD as occupied by PTH, but in contrast to a straight, continuous PTH helix, the PTHrP helix is gently curved and C-terminally “unwound.” The receptor accommodates the altered binding modes by shifting the side chain conformations of two residues within the binding groove: Leu-41 and Ile-115, the former acting as a rotamer toggle switch to accommodate PTH/PTHrP sequence divergence, and the latter adapting to the PTHrP curvature. Binding studies performed with PTH/PTHrP hybrid ligands having reciprocal exchanges of residues involved in different contacts confirmed functional consequences for the altered interactions and enabled the design of altered PTH and PTHrP peptides that adopt the ECD-binding mode of the opposite peptide. Hybrid peptides that bound the ECD poorly were selective for the G protein-coupled PTH1R conformation. These results establish a molecular model for better understanding of how two biologically distinct ligands can act through a single receptor and provide a template for designing better PTH/PTHrP therapeutics.The parathyroid hormone receptor (PTH1R)³ is a class B G protein-coupled receptor (GPCR) that transduces signals from two related signaling molecules that have distinct functions in biology: parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) (Ref. 1; reviewed in Ref. 2). PTH is an 84-amino acid polypeptide endocrine hormone that is produced by the parathyroid glands and secreted into the circulation in response to low calcium levels (reviewed in Refs. 3–5), to act on bone and kidney cells and thus restore blood calcium to normal levels. In bone, PTH directly stimulates osteoblasts, resulting in bone formation (reviewed in Ref. 6), which in turn activate osteoclasts to induce bone resorption. In the kidney, PTH stimulates the reabsorption of filtered calcium, inhibits the reabsorption of phosphate, and stimulates the synthesis of 1,25-dihydroxyvitamin D3. The paradoxical anabolic/catabolic actions of PTH on bone can be modulated by exogenous PTH, and provide the molecular basis for the clinical use of PTH as an anabolic therapy for osteoporosis (7). Anabolic PTH therapy requires intermittent administration to minimize bone-resorptive effects, which predominate with sustained administration of PTH. PTHrP is a 141-amino acid polypeptide that was originally isolated as the factor responsible for humoral hypercalcemia of malignancy (8–11) and was subsequently shown to be a critical developmental paracrine factor that controls endochondral bone formation (Refs. 12, 13; reviewed in Ref. 14). PTHrP can also mediate bone-anabolic effects when administered to osteoporosis patients (15) and has been suggested to be more anabolic than PTH due to a differential effect on the coupled bone formation and resorptive responses (16).PTH and PTHrP are encoded by separate genes, each of which is found in vertebrate species ranging from fish to man. How PTH and PTHrP evolved to mediate distinct biological activities: calcium/phosphate homeostasis and tissue development, respectively, via actions upon a single receptor, remains unclear. Amino acid sequence homology is most apparent in the first 34-residue segments of the proteins, and N-terminal 34-residue peptide fragments of PTH and PTHrP are sufficient for high affinity binding to the PTH1R and are generally found to be equally potent for stimulating cAMP formation in PTH1R-expressing cells (1). The interaction of the (1–34)-length ligand with the PTH1R has been postulated to follow a “two-domain” model: residues within the approximate (1–14) segment interact with the 7-transmembrane (7-TM) helical domain embedded in the membrane, and residues within the approximate (15–34) segment interact with the N-terminal extracellular domain (ECD) of the receptor (17, 18). The 1–14 domains of PTH and PTHrP share eight amino acid sequence identities, reflecting a critical role in activating the receptor (18), while the 15–34 domains share only three amino acid identities, despite a critical role in imparting high affinity binding to the receptor.Recent studies suggest that PTH and PTHrP differ in their relative capacities to bind to two pharmacologically distinguishable high-affinity PTH1R conformations (19–22). One conformation, termed R⁰, is stable in the presence of GTPγS, but presumably in the absence of G protein coupling, correlates with prolonged signaling responses in vitro and in vivo, and is bound preferentially by PTH-(1–34). The other conformation, termed RG, is sensitive to GTPγS addition, promoted by the overexpression of a high affinity variant of Gα_s, and bound preferentially by PTHrP-(1–36). A mechanistic basis for the differing capacities of PTH and PTHrP ligands to bind to these altered PTH1R conformations is not clear at present, although, both the (1–14) and (15–34) portions of PTH contribute importantly to the capacity to bind stably to the proposed R⁰ conformation (19, 21, 22).We previously developed a method that allowed us to determine the high resolution crystal structure of recombinant PTH1R ECD in complex with the 15–34 synthetic fragment of PTH (23). The PTH1R ECD adopts a tertiary fold that is conserved among class B GPCR ECDs (24–26), and the PTH(15–34)NH₂ domain binds as a continuous and straight amphipathic α-helix to a hydrophobic groove in the ECD. Here we present the high resolution crystal structure of the PTHrP 12–34 fragment in complex with the PTH1R ECD, which reveals a distinct docking conformation toward the C terminus of the PTHrP peptide. Based on the structural differences, we designed hybrid PTH/PTHrP peptides exchanged for residues involved in altered ECD contacts; functional analyses of these peptides confirmed that the altered modes of binding indeed translate into functional consequences in terms of receptor affinity. These results provide critical insights into how PTH and PTHrP can act through a single receptor, and a structural model for designing better PTH/PTHrP analogs for treating osteoporosis.     

Identification of Peptides That Mimic the Pertussis Toxin Binding Site on Bovine Fetuin

The introduction of acellular pertussis vaccines has greatly enhanced the safety profile of vaccines to prevent whooping cough. Pertussis toxin (Ptx) is one component produced by Bordetella pertussis that is contained in all of these vaccines, either in combination with other known pertussis virulence factors or as the sole pertussis component, combined with tetanus and diphtheria toxoids. A hydrogen peroxide toxoid of Ptx has been shown to be efficacious in preventing pertussis infections in a mass vaccination trial and is presently licensed in the United States and Europe (B. Trollfors, J. Taranger, T. Lagergard, L. Lind, V. Sundh, G. Zackrisson, C. U. Lowe, W. Blackwelder, and J. B. Robbins, N. Engl. J. Med. 333:1045-1050, 1995). The industrial production of Ptx can be performed through the cultivation of B. pertussis in well-defined growth media, in which the components can be well characterized and their origins can be documented. Once the bacteria are removed from the culture, Ptx can be isolated from the supernatant and purified by using the technique described by Sekura et al. (R. D. Sekura, F. Fish, C. R. Manclark, B. Meade, and Y. L. Zhang, J. Biol. Chem. 258:14647-14651, 1983). The only drawback of this procedure, which combines two affinity chromatography steps, one with Blue Sepharose and a second with matrix-bound bovine fetuin (BF), is the source and purity of the BF. Concern about vaccine preparations that may possibly risk contamination by material associated with bovine spongioform encephalopathy has continued to increase. We thus sought a replacement for the BF affinity chromatography and, more specifically, for the glycosidic moiety on BF. We describe here the identification of a seven-amino-acid peptide that mimics the glycosidic moiety on BF to which Ptx binds. Furthermore, we have constructed an affinity column containing this peptide that can be used to replace BF in Ptx purification. Finally, we used the X-ray crystallographic structure of Ptx bound to the oligosaccharide moiety of BF as a scaffold and replaced the oligosaccharide with the peptide.