验证性筛选PDZ结构域结合配体文库快速研究HtrA2/Omi PDZ结构域的结合特性

HtrA2/Omi是一种线粒体丝氨酸蛋白酶, 在哺乳动物细胞中具有双重功能, 即诱导细胞凋亡和参与维持线粒体活性的动态平衡. PDZ结构域是最重要的蛋白质相互作用结构域之一, 参与多种生物学过程, 如细胞信号转导、蛋白质降解、细胞骨架组织等. 最近研究发现, HtrA2/Omi蛋白的PDZ结构域与配体的相互作用, 可以调节HtrA2/Omi蛋白自身的水解酶活性.以HtrA2/Omi PDZ结构域为研究对象, 用酵母双杂交系统验证性筛选PDZ结构域结合配体文库, 快速研究该结构域的结合特性, 并在人类全蛋白质组范围内预测并发现该结构域新的相互作用蛋白, 最后分析这些新的相互作用所能够形成的最小相互作用网络来评估其可信度. 研究结果揭示了HtrA2/Omi PDZ结构域新的结合特性, 即: 不仅能够结合已报道的II类PDZ配体而且还可以结合I类和III类PDZ配体, 并且配体-3位氨基酸具有一定范围内的可变性. 而且, 发现了7个新的HtrA2/Omi PDZ结构域相互作用蛋白, 为进一步阐明HtrA2/Omi蛋白的生物学功能提供了重要线索. 同时证明了验证性筛选目的结构域结合配体文库, 这一结构域结合特性研究新策略的实用性和高效性.     

利用随机多肽文库研究ZO-1中PDZ3结构域的配体结合特点

建立一种研究PDZ结构域配体结合特点的简单方法 .利用酵母双杂交技术从随机多肽文库中寻找所有可能与ZO 1中PDZ3结构域结合的C末端序列 ,从现有蛋白质数据库中检索所有具有该C末端蛋白 .利用液体培养物 β 半乳糖苷酶检测实验 ,比较文库中筛选的C末端序列和已知的PDZ3结构域结合配体———JAM的C末端 (SFLV)与PDZ3结构域结合的强弱 .共筛选到 3个阳性克隆 ,其C末端序列分别为 LGWV、 LVWV和 DEWV .前 2者属于第二类PDZ结构域 ,后者属于第三类 .蛋白质数据库检索结果表明 ,有多个蛋白质具有 LGWV、 LVWV末端 ,没有检索到任何具有 DEWV末端的蛋白质 .结合强度实验结果表明 ,它们与PDZ3结构域结合强度依次为 DEWV > LGWV > LVWV > SFLV ,说明筛选的 3个C末端除了反映ZO 1中PDZ3结构域可能的潜在结合配体外 ,也有可能成为JAM蛋白阻断性试剂甚至药物的重要组成部分之一 .利用随机多肽文库 ,可以尽可能寻找所有可能与PDZ结构域结合的C末端序列 ,大大提高了基因文库筛选的效率     

酵母双杂交技术筛选并回转验证在胞内与PML-C结构域相互作用的蛋白

目的 利用酵母双杂交技术在活细胞内筛选并回转验证与PML-C结构域相互作用的蛋白质.方法 通过诱饵质粒pGBKT7-PML-C,利用酵母双杂交系统从白血病细胞cDNA文库中筛选与PML-C结构域相互作用的蛋白质.结果 利用酵母双杂交技术筛选到43个能与PML-C结构域相互作用的克隆;经进一步的归类与酵母回转试验得到9个阳性克隆.结论 在细胞内PML-C结构域能与多种蛋白质有相互作用.中性粒细胞弹性蛋白酶（neutrophil elastase,NE）介导的急性早幼粒细胞白血病的发生可能与这些相互作用所致的生物学功能改变有关.     

PDZ结构域介导的PIP脂质信号转导

PDZ结构域是调控蛋白质/蛋白质相互作用的一类重要结构域,能特异结合蛋白质C末端一段有规律的氨基酸序列。含有PDZ结构域的支架蛋白能够组装成超大的蛋白质复合体来调控细胞内的信号转导通路。最新研究表明,PDZ结构域还能与PIP脂质直接相互作用,从而参与调控PIP脂质信号通路。将综合最新研究进展,阐明PDZ结构域与PIP脂质的作用方式,以及对相关PIP脂质信号转导的调控过程。     

PDZ结构域的结构特点及其识别特异性配体的机制

PDZ结构域作为介导蛋白质之间相互作用的重要结构域之一,参与到细胞内运输、离子通道、以及各种信号传导通路等多种生物学过程.PDZ结构域是由80~100个氨基酸组成的小的球状结构域,对某些多PDZ结构域蛋白来说,需要一前一后形成串联体才能正确折叠.另外,PDZ结构域相互之间也可以形成同源或异源二聚体.这些PDZ结构域的突出特点是能特异性地识别配体靶蛋白C末端短的氨基酸序列,但有些也能识别靶蛋白的内部β发夹结构.而一些支架蛋白的PDZ结构域与细胞膜上脂类的相互作用则增加了其与膜的亲和性.本文简要概括了PDZ结构域的结构特点及其对配体的各种特异性识别的机制,从而为研究各种PDZ蛋白的功能提供了结构基础.     

Tec家族蛋白酪氨酸激酶Itk PH结构域与OS-9蛋白的相互作用

用酵母双杂交技术筛选与ItkPH结构域相互作用的蛋白分子 ,以了解Itk的功能及其在T细胞信号转导中的位置与作用 .Itk的PH结构域扩增后克隆入酵母双杂交系统的pLexA载体 ,转化酵母细胞EGY4 8(p8op lacZ) ,经检测PH结构域无自激活作用 ,且对酵母细胞无毒性作用 .用PH结构域作为“钓饵”蛋白 ,在酵母双杂交系统中筛选构建于AD载体的T细胞cDNA文库 .将PH结构域及筛库所得基因片段分别进行融合表达 ,用于体外结合实验 ,进一步证实二者的相互作用 .经营养缺陷选择、诱导筛选和鉴定确证 ,筛库所得的插段约 15 0 0bp的文库质粒为一真阳性克隆 .经blast比较分析为骨肉瘤、横纹肌肉瘤等肿瘤组织中高表达的os 9基因 .体外结合实验也表明 ,ItkPH结构域可与该基因表达产物结合 .Itk的PH结构域可与OS 9蛋白相互作用 .二者结合的意义有待进一步研究     

利用酵母双杂交技术筛选人PROKR2蛋白C端相互作用蛋白

目的:利用酵母双杂交技术从人c DNA文库中筛选人前动力蛋白受体2(hPROKR2)C端相互作用蛋白。方法:复苏酵母菌Y2HGold-hPROKR2-C,与人c DNA文库杂交,计算杂交效率;筛选相互作用克隆并测序,寻找hPROKR2蛋白C端相互作用蛋白;通过在DDO/X、QDO/A/X平皿上划线,对筛选到的人c DNA文库质粒的自活性进行验证;采用GST pull down进一步验证两者之间的相互作用。结果:酵母双杂交实验的杂交融合效率为1.375×10~8;在人cDNA文库中筛选出hPROKR2蛋白C端相互作用蛋白SNAPIN,GST pull down实验证实两者之间存在相互作用。结论:SNAPIN可与hPROKR2蛋白C端结合。     

PICK1：一个功能多样的药靶蛋白

蛋白质是生命功能的执行者.生命体中某些关键蛋白的功能异常往往是导致疾病发生的根本原因.这些疾病相关蛋白极有可能成为药物靶点,为新药研发和疾病治疗提供重要线索. PICK1蛋白（protein interacting with Cα kinase 1）结合能力广泛、功能多样以及在多种重要疾病（如：癌症、精神分裂症、疼痛、帕金森综合症等）的发生发展过程中发挥潜在的作用,使其成为一个可能的药靶蛋白. PICK1与绝大多数配体蛋白的相互作用是通过其PDZ结构域与配体C末端区域的结合介导的,使PICK1的PDZ结构域成为一个潜在的药物靶点.因此,可以利用生物小分子物质特异性地结合PICK1的PDZ结构域,干扰或阻断PICK1与配体蛋白的天然相互作用,最终达到治疗相关疾病的目的.     

转录因子X-box结合蛋白1相互作用蛋白的筛选和鉴定

X-box 结合蛋白 1 是一种重要的转录因子,参与体内多项信号转导过程. 为进一步研究 XBP1 的生物学功能,运用酵母双杂交技术在肝细胞文库中筛选 XBP1 的结合蛋白. 首先运用 PCR 技术扩增获得 XBP1 的编码序列,克隆至 pGEM-T 载体,经测序鉴定后,亚克隆至诱饵载体 pGBKT7 中,转化酵母 AH109(a type). 免疫印迹检测诱饵质粒 pGBKT7-XBP1 在AH109 酵母中的表达之后,含有诱饵质粒的酵母 AH109 与含有肝细胞 cDNA 文库质粒 pACT2 的酵母 Y187(αtype)配合,配合后的二倍体酵母生长在含有 X-α-gal 的营养缺陷型培养基上 (SD/-Trp-Leu-His-Ade) 进行选择和筛选,经测序和序列比对确定阳性克隆的开放读码框 ORF,得到 7 种不同的蛋白质. 为了进一步验证这些筛选蛋白质与 XBP1 的相互作用,克隆其中一种蛋白质 MT1E,并运用 GST pulldown 和免疫共沉淀技术成功检测了 MT1E 和 XBP1 的相互作用(体外 / 体内),结果提示,MT1E 可能是 XBP1 的一个新的调节蛋白. 通过酵母双杂交技术筛选得到的 7 种蛋白质分别与肝细胞基础代谢、蛋白质的合成与运输、细胞的增殖与凋亡密切相关. 上述结果有助于揭示 XBP1 的生物学功能,为进一步探讨 XBP1 的表达和调控机制提供新线索.     

与雌激素受体β相互作用的蛋白的分离及鉴定

目的:从乳腺文库中分离与雌激素受体β(ERβ)相互作用的蛋白质。方法:将ERβ基因克隆入酵母表达载体pGBKT7中,利用酵母双杂交筛选技术,从乳腺文库中分离与ERβ相互作用的蛋白质。结果:分离到与ERβ相互作用的蛋白质FBLN1,并进一步确定了ERβ与FBLN1作用的结构域为AF1及DBD结构域。结论:FBLN1可能参与ERβ信号途径并共同调节细胞的增殖。ERβ与FBLN1相互作用的发现为进一步研究ERβ与FBLN1在生理及病理中的作用奠定了基础。     

Comparative structural analysis of the Erbin PDZ domain and the first PDZ domain of ZO-1. Insights into determinants of PDZ domain specificity

We report a structural comparison of the first PDZ domain of ZO-1 (ZO1-PDZ1) and the PDZ domain of Erbin (Erbin-PDZ). Although the binding profile of Erbin-PDZ is extremely specific ([D/E][T/S]WV(COOH)), that of ZO1-PDZ1 is similar ([R/K/S/T][T/S][W/Y][V/I/L](COOH)) but broadened by increased promiscuity for three of the last four ligand residues. Consequently, the biological function of ZO-1 is also broadened, as it interacts with both tight and adherens junction proteins, whereas Erbin is restricted to adherens junctions. Structural analyses reveal that the differences in specificity can be accounted for by two key differences in primary sequence. A reduction in the size of the hydrophobic residue at the base of the site(0) pocket enables ZO1-PDZ1 to accommodate larger C-terminal residues. A single additional difference alters the specificity of both site(-1) and site(-3). In ZO1-PDZ1, an Asp residue makes favorable interactions with both Tyr(-1) and Lys/Arg(-3). In contrast, Erbin-PDZ contains an Arg at the equivalent position, and this side chain cannot accommodate either Tyr(-1) or Lys/Arg(-3) but, instead, interacts favorably with Glu/Asp(-3). We propose a model for ligand recognition that accounts for interactions extending across the entire binding site but that highlights several key specificity switches within the PDZ domain fold.     

High‐energy water sites determine peptide binding affinity and specificity of PDZ domains

PDZ domains have well known binding preferences for distinct C‐terminal peptide motifs. For most PDZ domains, these motifs are of the form [S/T]‐W‐[I/L/V]. Although the preference for S/T has been explained by a specific hydrogen bond interaction with a histidine in the PDZ domain and the (I/L/V) is buried in a hydrophobic pocket, the mechanism for Trp specificity at the second to last position has thus far remained unknown. Here, we apply a method to compute the free energies of explicit water molecules and predict that potency gained by Trp binding is due to a favorable release of high‐energy water molecules into bulk. The affinities of a series of peptides for both wild‐type and mutant forms of the PDZ domain of Erbin correlate very well with the computed free energy of binding of displaced waters, suggesting a direct relationship between water displacement and peptide affinity. Finally, we show a correlation between the magnitude of the displaced water free energy and the degree of Trp‐sensitivity among subtypes of the HTRA PDZ family, indicating a water‐mediated mechanism for specificity of peptide binding.     

The complete amino acid sequence of the low molecular weight cytosolic acid phosphatase

This paper presents the complete amino acid sequence of the low molecular weight acid phosphatase from bovine liver. This isoenzyme of the acid phosphatase family is located in the cytosol, is not inhibited by L-(+)-tartrate and fluoride ions, but is inhibited by sulfhydryl reagents. The enzyme consists of 157 amino acid residues, has an acetylated NH2 terminus, and has arginine as the COOH-terminal residue. All 8 half-cystine residues are in the free thiol form. The molecular weight calculated from the sequence is 17,953. The sequence was determined by characterizing the peptides purified by reverse-phase high performance liquid chromatography from tryptic, thermolytic, peptic, Staphylococcus aureus protease, and chymotryptic digests of the carboxymethylated protein. No sequence homologies were found with the two known acylphosphatase isoenzymes or the metalloproteins porcine uteroferrin and purple acid phosphatase from bovine spleen (both of which have acid phosphatase activity). Two half-cystines at or near the active site were identified through the reaction of the enzyme with [14C] iodoacetate in the presence or in the absence of a competitive inhibitor (i.e. inorganic phosphate). Ac-A E Q V T K S V L F V C L G N I C R S P I A E A V F R K L V T D Q N I S D N W V I D S G A V S D W N V G R S P N P R A V S C L R N H G I N T A H K A R Q V T K E D F V T F D Y I L C M D E S N L R D L N R K S N Q V K N C R A K I E L L G S Y D P Q K Q L I I E D P Y Y G N D A D F E T V Y Q Q C V R C C R A F L E K V R-OH.     

A C-terminal class I PDZ binding motif of EspI/NleA modulates the virulence of attaching and effacing Escherichia coli and Citrobacter rodentium

Enteropathogenic Escherichia coli induces characteristic attaching–effacing (A/E) lesions on the intestinal mucosa during infection. The locus of enterocyte effacement is essential for A/E lesion formation and encodes a type III secretion system that translocates multiple effector proteins into the host cell. Following translocation, EspI/NleA localizes to the Golgi. Using the yeast two-hybrid system (Y2HS) and PSD-95/Disk-large/ZO-1 (PDZ)-domain protein array overlays, we identified 15 putative host-interacting partners of EspI. All but two of the target proteins contained PDZ domains. Examination of the EspI amino acid sequence revealed a C-terminal consensus class I PDZ binding motif. Deletion of the last 7 amino acids of EspI to generate EspI_ΔC7 abrogated the Y2HS interaction between EspI and 5 of the 6 putative host cell target proteins tested. Deletion of the EspI PDZ binding motif also resulted in delayed trafficking of EspI to the Golgi. Using a mouse model of infection, we showed that Citrobacter rodentium expressing truncated EspI_ΔC7 was attenuated when in competition with C. rodentium expressing full-length EspI. Overall, these results suggested that EspI may modulate the virulence of A/E pathogens by binding host PDZ-domain proteins.     

SH2 domains from suppressor of cytokine signaling-3 and protein tyrosine phosphatase SHP-2 have similar binding specificities

Suppressor of cytokine signaling-3 (SOCS-3) and the protein tyrosine phosphatase SHP-2 both regulate signaling by cytokines of the interleukin-6 family, and this is dependent upon recruitment to tyrosine 757 in the shared cytokine receptor subunit gp130. To better explore the overlap in ligand binding specificities exhibited by these two signaling regulators, we have mapped the phosphopeptide binding preferences of the SH2 domains from SOCS-3 and SHP-2. Degenerate phosphopeptide libraries were screened against recombinantly produced SH2 domains to determine the sequences of optimal phosphopeptide ligands. We found that the consensus ligand binding motif for SOCS-3 was pY-(S/A/V/Y/F)-hydrophobic-(V/I/L)-hydrophobic-(H/V/I/Y), while the consensus motif for SHP-2 was pY-(S/T/A/V/I)-X-(V/I/L)-X-(W/F). We validated these data through the design of phosphopeptide ligands based on the consensus motifs and found that these bound to SOCS-3 and SHP-2 with high affinity. Finally, we have compared the affinity of SOCS-3 for binding to phosphopeptides representing putative docking sites in the gp130, leptin and erythropoietin receptors. While SOCS-3 binds with much higher affinity to a gp130 phosphopeptide than to phosphopeptides derived from the other receptors, multiple SOCS-3 binding sites are predicted to exist in the leptin and erythropoietin receptors which may compensate for weaker binding to individual sites.     

The complete amino acid sequence of coagulogen isolated from Southeast Asian horseshoe crab, Carcinoscorpius rotundicauda

The complete amino acid sequence of coagulogen purified from the hemocytes of the horseshoe crab Carcinoscorpius rotundicauda was determined by characterization of the NH2-terminal sequence and the peptides generated after digestion of the protein with lysyl endopeptidase, Staphylococcal aureus protease V8 and trypsin. Upon sequencing the peptides by the automated Edman method, the following sequence was obtained: A D T N A P L C L C D E P G I L G R N Q L V T P E V K E K I E K A V E A V A E E S G V S G R G F S L F S H H P V F R E C G K Y E C R T V R P E H T R C Y N F P P F V H F T S E C P V S T R D C E P V F G Y T V A G E F R V I V Q A P R A G F R Q C V W Q H K C R Y G S N N C G F S G R C T Q Q R S V V R L V T Y N L E K D G F L C E S F R T C C G C P C R N Y Carcinoscorpius coagulogen consists of a single polypeptide chain with a total of 175 amino acid residues and a calculated molecular weight of 19,675. The secondary structure calculated by the method of Chou and Fasman reveals the presence of an alpha-helix region in the peptide C segment (residue Nos. 19 to 46), which is released during the proteolytic conversion of coagulogen to coagulin gel. The beta-sheet structure and the 16 half-cystines found in the molecule appear to yield a compact protein stable to acid and heat. The amino acid sequences of coagulogen of four species of limulus have been compared and the interspecies evolutionary differences are discussed.     

Identification of the hydrophobic ligand binding pocket of the S1P1 receptor

Sphingosine 1-phosphate (S1P), a naturally occurring sphingolipid mediator and also a second messenger with growth factor-like actions in almost every cell type, is an endogenous ligand of five G protein-coupled receptors (GPCRs) in the endothelial differentiation gene family. The lack of GPCR crystal structures sets serious limitations to rational drug design and in silico searches for subtype-selective ligands. Here we report on the experimental validation of a computational model of the ligand binding pocket of the S1P1 GPCR surrounding the aliphatic portion of S1P. The extensive mutagenesis-based validation confirmed 18 residues lining the hydrophobic ligand binding pocket, which, combined with the previously validated three head group-interacting residues, now complete the mapping of the S1P ligand recognition site. We identified six mutants (L3.43G/L3.44G, L3.43E/L3.44E, L5.52A, F5.48G, V6.40L, and F6.44G) that maintained wild type [32P]S1P binding with abolished ligand-dependent activation by S1P. These data suggest a role for these amino acids in the conformational transition of S1P1 to its activated state. Three aromatic mutations (F5.48Y, F6.44G, and W6.48A) result in differential activation, by S1P or SEW2871, indicating that structural differences between the two agonists can partially compensate for differences in the amino acid side chain. The now validated ligand binding pocket provided us with a pharmacophore model, which was used for in silico screening of the NCI, National Institutes of Health, Developmental Therapeutics chemical library, leading to the identification of two novel nonlipid agonists of S1P1.     

正颤蚓的生长发育及繁殖生物学的研究

在实验室饲养的条件下,研究了正颤蚓在15℃、20℃、25℃水温的生长发育和繁殖过程。结果表明正颤蚓从其受精卵在蚓茧内发育,孵出生长到产卵,在15℃水温需100—123d,在20℃水温需70—90d,在25℃水温需48—64d。蚓自蚓茧内孵出后的165d生长期内,其15℃、20℃、25℃水温的体重生长回归方程分别是W=6.87/［1+136.48     

Exploring the cause of drug resistance by the detrimental missense mutations in KIT receptor: computational approach

In this work, we computationally identified the most detrimental missense mutations of KIT receptor causing gastrointestinal stromal tumors and analyzed the drug resistance of these missense mutations. Out of 31 missense mutations, 19 variants were commonly found less stable, deleterious and damaging by I-Mutant 2.0, SIFT and PolyPhen programs, respectively. Subsequently, we performed modeling of these 19 variants to understand their change in conformations with respect to native KIT receptor by computing their RMSD. Further, the native and 19 mutants were docked with the drug ‘Imatinib’ to explain the drug resistance of these detrimental missense mutations. Among the 19 mutants, we found by docking studies that 12 mutants, namely, F584C, F584L, V654A, L656P, T670I, R804W, D816F, D816V, D816Y, N822K, Y823D and E839K had less binding affinity with Imatinib than the native type. Finally, we analyzed that the loss of binding affinity of these 12 mutants, was due to altered flexibility in their binding amino acids with Imatinib as compared with native type by normal mode analysis. In our work, we found the novel data that the majority of the drug-binding amino acids in those 12 mutants had encountered loss of flexibility, which could be the theoretical basis for the cause of drug insensitivity.