t-PA突变体的构建、表达及生物学特性分析

t-PA是纤溶系统的生理性激活剂,但它在血浆中半衰期甚短及活性可被天然快速抑制剂PAI-1抑制等生理特性成为其作为临床溶栓剂的主要缺陷。为了研制新型t-PA溶栓剂,本文根据t-PA结构与功能研究的最新信息,针对延长t-PA半衰期、提高特异活性及引入PAI-1抗性等,设计并构建了8种t-PA突变体。进而在COS-7及CHO细胞中进行了表达研究,并对突变体表达产物的特性进行了分析和评价。     

人组织型纤溶酶原激活剂（t－PA）突变体FrGGI的构建、表达及特性分析

为了获得半衰期延长,特异活性提高及具有ＰＡＬ－１抗性的新型ｔ－ＰＡ溶栓剂,利用基因重组及定位突变技术构建了ｔ－ＰＡ的Ｋ１、Ｋ２区糖基化位点消除,ＰＡＩ－１结合位点缺失,Ｆ与Ｅ区连接序列Ｈｉｓ４４～Ｓｅｒ５０置换为纤粘蛋白Ⅰ型Ｆ区间连接序列ＧｌｕＳｅｒＬｙｓＰｒｏＧｌｕＡｌａＧｌｕＧｌｕ的ｔ－ＰＡ组合突变体ＦｒＧＧＩ,并在中国仓鼠卵巢细胞中获得了高效表达。对表达产物的生物学特性分析表明,ＦｒＧＧＩ在大鼠血浆中的半衰期延长了１５倍,并获得了ＰＡＩ－１抗性,是一株很有希望的新型溶栓剂候选株。     

半衰期延长获得PAI－1抗性的t—PA突变体的构建、表达及特性分析

用基因重组及定位突变技术成功地构建了t·PA的Kl区缺失突变体t—PAdelKl、PAI-1结合位点缺失突变体t—PA del(296—302)及两者的组合突变体t—PA del(Kl·296—302),并在COS－7细胞中实现三者的暂时性表达,在cHO细胞中实现了t·PA deI(K1．296—302)的稳定性表达．对表达产物的生物学特性分析表明,t—PA del(296—302)及t-PA del(K1,296—302)获得了Pal－1抗性．因时t-PA del(K1．296—302)在大鼠体内的半衰期延长约5倍,而与纤维蛋白的亲和力只略有下降。因此,此组合突变体有可能成为优于野生t—PA的新型溶栓剂候选株．     

人组织型纤溶酶原激活剂（t—PA）突变体FrGGI的构建,表达及特性…

为了获得半衰期延长,特异活性提高及具有ＰＡＩ－１抗性的新型ｔ－ＰＡ溶性剂,利用基因重组及定位突变技术构建了ｔ－ＰＡ的Ｋ１、Ｋ２区糖基化位点消除,ＰＡＩ－１结合位点缺失,Ｆ与Ｅ区连接序列Ｈｉｓ４４￣Ｓｅｒ５０置换为纤粘蛋白Ｉ型Ｆ区间连接序列Ｇｌｕ Ｓｅｒ Ｌｙｓ Ｐｒｏ Ｇｌｕ Ａｌａ Ｇｌｕ Ｇｌｕ的ｔ－ＰＡ组合突变体ＦｒＧＧＩ,并在中国仓鼠卵巢细胞中获得了高效表达,对表达产物的生物学特性分析表明     

t-PA F-E复合片段的三维结构模建及其结构与功能关系的研究

组织型纤溶酶原激活剂(t-PA)是一种在血栓和心梗的临床治疗中具有巨大应用前景的蛋白分子。野生型t-PA由于半衰期短,用量大,在实际治疗过程中会产生一些副作用,因此研制半衰期适当延长、血栓特异性增强的新型t-PA突变体非常重要。大量实验证明F区的C末端或F-E连接区对t-PA在血液中的半衰期及其血栓特性发挥了重要作用。1994年Smith等人报道了利用NMR研究F区-E区复合片     

组织型纤溶酶原激活剂（t—PA）工程细胞系遗传特性及成瘤性分析

组织型纤溶酶原激活剂(t-PA)因其在防止血栓形成中起重要作用而受到人们的重视。但由于t-PA在血液中半衰期很短,作为溶栓药,一时难于推广。为了延长半衰期、增强其特异活性,本组构建了t-PA突变体并在CHO-dhfr~-细胞中获得了高效表达。我们在细胞培养基中加入秋水仙素,通过低张、固定、染色,进行染色体分析,结果表明,t-PA工程细胞系染色体条数为20条,畸变类型有异着丝粒。四倍体、裂隙、断片,畸变率为15％,属于正常范围。同时我们对该细胞系进行成瘤性试验,选用4周龄裸鼠作为试验鼠,以Hela细胞为阳性对照,CHO-dhfr~-细胞为阴性对照,试验表明:t-PA工程细胞及表达产物对裸鼠均无成瘤性。     

抗t-PA单克隆抗体的制备及初步应用

用猪心t-PA和人黑色素瘤细胞分泌的t-PA作抗原,经腹腔及脾内免疫Bal b/c小鼠。用细胞融合技术制备杂交瘤细胞,细胞融合串达85％,获得4株抗t-PA杂交瘤细胞株,鼠腹水抗体效价达1∶10~5;Western Blot结果表明该单抗所结合的抗原分子量与t-PA相符,证明所获得的单抗为特异的抗t-PA单抗;对t-PA的纤溶活性中和抑制试验结果表明,4株单抗中的1株能完全抑制st-PA的纤溶活性,另外3株表现出程度不等的抑制;其中1株亚类为IgG,另外3株为IgM。杂交瘤细胞株无支原体污染;染色体数目正常。对该抗体进行初步纯化后,将其应用于rt-PA的研究中。     

t-PA昆虫细胞表达及特性分析

应用RT-PCR技术,从人黑色素瘤Bowes细胞株中扩增出人组织型纤溶酶原激活剂(tissue-typeplasminogenactivator,t-PA)cDNA。序列分析证实,与国外的报道完全一致。将含完整阅读框架的人t-PAcDNA克隆至昆虫细胞表达转移载体pBacPAK8中,获得重组质粒pBac-tPA。应用脂质体共转染法,将pBactPA和线性化杆状病毒BacPAK6DNA共转染Tn-5B-1昆虫细胞。经空斑筛选获得11株重组病毒。经PCR鉴定与生物活性测定,获得一株高效表达t-PA的重组病毒BactPA3。在含胎牛血清的培养基中,t-PA表达活性在感染(MOI≈10)后72h左右达到最高,为3.04×103IU／ml,即相当于1.8×104IU／106细胞;在无血清培养基中,t-PA最高表达水平相差不大,但时间为感染后132h左右。经SDS-PAGE纤维蛋白自显影分析,分子量为68kda左右。与从人黑色素瘤细胞培养液中提纯的天然t-PA相比,其受纤维蛋白原激活的特性、和纤维蛋白的亲和力及在血浆中的失活速率基本相同。表达的t-PA在血液循环中的半衰期为7min。     

重组人纤溶酶原激活剂转基因兔的制备及其表达产物的检测

人组织纤溶酶原激活剂(ht-PA)是临床血栓疾病治疗的主要生物药品,应用转基因动物乳腺生物反应器制备已有报道,但重组人纤溶酶原激活剂(rh PA)在转基因动物中表达尚未见报道。设计了以人t-PA的F、E、K1区位点缺失体为编码序列,以山羊β-酪蛋白/CMV杂合启动/增强子,构建了乳腺特异性表达载体PCL25/rh PA,通过受精卵原核显微注射法制备转基因兔。用PCR检测及其产物测序筛选转基因兔,ELISA试剂盒及纤维蛋白平板溶圈法检测转基因兔的表达产物。实验获得原代转基因兔5只(2♂,3♀),其中2只乳腺中表达人重组纤溶酶原激活剂,rh PA浓度最高可达400μg/m L,表达产物比活性是现有阿替普酶的150-200倍。实验结果表明,重组人纤溶酶原激活剂(rh PA)可以在山羊β-酪蛋白/CMV杂合启动/增强子调控下获得乳腺特异性表达,表达重组蛋白具有较高的溶栓活性。     

人肾小球系膜细胞纤溶酶原激活物抑制物cDNA克隆和高效表达

利用逆转录 聚合酶链式反应 (RT- PCR)方法 ,从中国正常人肾小球系膜细胞总RNA中扩增出人纤溶酶原激活物抑制物 (PAI 1 )基因cDNA编码区序列 ,并定向亚克隆至pUC1 9质粒 ,克隆的PAI -1cDNA去除了信号肽核苷酸序列并加入新的起始密码ATG ,编码区序列与文献报道的人内皮细胞PAI -1cDNA序列完全相同 .将PAI -1cDNA定向亚克隆至原核表达质粒 pBV2 2 0 ,构建了重组PAI -1基因表达质粒pBV2 2 0 PAI -1 ,在大肠杆菌中得到了高效表达 ,重组PAI -1蛋白表达占菌体总蛋白 45 % .Westernblotting检测 ,在分子量约为 43.0ku处出现一特异性蛋白质条带 .对形成包涵体的表达产物进行变复性处理及FPLC纯化 ,获得纯度 97%以上的潜伏态重组PAI -1 .经 4mol/L盐酸胍激活后 ,重组PAI- 1具有与天然PAI- 1同样的生物学活性 ,对尿激酶型纤溶酶原激活物 (u- PA)具有显著抑制活性 .     

Registration of the rod is not critical for the phosphorylation-dependent regulation of smooth muscle myosin.

A recent report has suggested that the interaction between the head and the rod region of smooth muscle myosin at S2 is important for the phosphorylation-mediated regulation of myosin motor activity [Trybus, K. M., Freyzon, Y., Faust, L. Z., and Sweeney, H. L. (1997) Proc. Natl. Acad. Sci. U.S.A. 74, 48-52]. To investigate whether specific amino acid residues at S2 or whether the registration of the 7-residue/28-residue repeat appearing in the alpha-helical coiled-coil structure of the rod are critical for such an interaction, two smooth muscle myosin mutants were constructed in which the N-terminal sequences of S2 were deleted to various extents. One mutant contained a deletion of 71 residues at the position immediately C-terminal to the invariant proline (Pro849) linking the S1 domain directly to the downstream sequence of the rod, while in another mutant, 53 residues were deleted at a position 56 residues downstream of Pro849. Despite these alterations which change the registration of both the 28-residue repeat and the 7-residue repeat found in myosin rod sequence, both myosin mutants showed a stable double-headed structure by electron microscopic observation. Both the actin-activated ATPase activity and the actin translocating activity of the mutants were completely regulated by the phosphorylation of the regulatory light chain. The actin sliding velocity of the two mutant myosins was the same as the wild-type recombinant myosin. Furthermore, the head configuration critical for myosin filament formation (extended or folded) was unchanged in either mutant. These results indicate that neither the specific amino acid residues nor the registration of the amino acid repeat in S2 is critical for the head configuration. These results indicate that neither a specific amino acid sequence at the head-rod junction nor the rod sequence registration is critical for the regulation of smooth muscle myosin.     

Synthesis and DNA-binding ability of Sp1 protein zinc finger domain and its peptidomimetics

The second zinc finger fragment of Sp1 (Sp1-ZF2), its mutant (Sp1-ZF2/HT. E²⁰ → H, R²³ → T), and two mimic analogues (ZF20 and ZF15) were synthesized by stepwise solid phase technique. The CD spectra and UV-visible spectrum with CoC1₂ indicated that the formation of zinc finger structure was affected not only by the hydrophobic amino acids but also by the change of the distance between Cys and His. Gel-retardat ion electrophoresis assays indicated that the Glu and Arg residues are very important for recognition. A single zinc finger like Sp1-ZF2 is able to bind DNA sequence specifically.     

Synthesis and DNA-binding ability of Spl protein zinc finger domain and its peptidomimetics

The second zinc finger fragment of Sp1 (Spl-ZF2), its mutant (Spl-ZF2/HT. E20→H, R23→T), and two mimic analogues (ZF20 and ZF15) were synthesized by stepwise solid phase technique. The CD spectra and UV-visible spectrum with CoCl2 indicated that the formation of zinc finger structure was affected not only by the hy-drophobic amino acids but also by the change of the distance between Cys and His. Gel-retardation electrophoresis as-says indicated that the Grlu and Arg residues are very important for recognition. A single zinc finger like Spl-ZF2 isable to bind DNA sequence specifically.     

Biochemical and functional characterization of human tissue-type plasminogen activator variants obtained by deletion and/or duplication of structural/functional domains

Five cDNA encoding human tissue-type plasminogen activator (t-PA) variants with deletion and/or duplication of structural/functional domains were cloned and expressed in Chinese hamster ovary cells. The mutants included: rt-PA-delta FE (where r represents recombinant), with deletion of the finger (F) and growth factor (E) domains; rt-PA-delta K1 delta K2, with replacement of kringle 1 (K1) by a second copy of kringle 2 (K2); and rt-PA-delta FK1 delta K2, rt-PA-delta EK1 delta K2, and rt-PA-delta FEK1 delta K2, with deletions in rt-PA-delta K1 delta K2 of the finger or growth factor domain or both, respectively. The variant rt-PAs, purified to homogeneity, were obtained essentially as single-chain molecules. CNBr-digested fibrinogen enhanced plasminogen activation between 110-fold with rt-PA-delta EK1 delta K2 and 150-fold with rt-PA-delta FEK1 delta K2 as compared to 140-fold with rt-PA. All rt-PA moieties showed a comparable concentration-dependent binding to fibrin, except rt-PA-delta FE, which had significantly reduced binding that was, however, partially restored by additional replacement of K1 with K2. All the rt-PA variants with two copies of K2 showed increased binding to lysine-Sepharose as compared to rt-PA, whereas rt-PA-delta FE had reduced binding. All rt-PA moieties induced a similar time- and concentration-dependent lysis of a 125I-fibrin-labeled plasma clot immersed in human plasma. Equally effective concentrations (causing 50% clot lysis in 2 h) ranged between 1.0 microgram/ml for rt-PA-delta K1 delta K2 and 1.6 micrograms/ml for rt-PA-delta FE as compared to 0.5 microgram/ml for rt-PA. Thus, replacement in rt-PA of K1 by a second copy of K2, which is known to contain a lysine-binding site, significantly enhances its affinity for lysine, with maintenance of its affinity for intact fibrin. Deletion of the finger and growth factor domains results in decreased fibrin affinity and fibrinolytic potency in a plasma milieu, which are partially restored by replacement of K1 by K2.     

Histone H3K4me3 binding is required for the DNA repair and apoptotic activities of ING1 tumor suppressor

Inhibitor of growth 1 (ING1) is implicated in oncogenesis, DNA damage repair, and apoptosis. Mutations within the ING1 gene and altered expression levels of ING1 are found in multiple human cancers. Here, we show that both DNA repair and apoptotic activities of ING1 require the interaction of the C-terminal plant homeodomain (PHD) finger with histone H3 trimethylated at Lys4 (H3K4me3). The ING1 PHD finger recognizes methylated H3K4 but not other histone modifications as revealed by the peptide microarrays. The molecular mechanism of the histone recognition is elucidated based on a 2.1 Å-resolution crystal structure of the PHD-H3K4me3 complex. The K4me3 occupies a deep hydrophobic pocket formed by the conserved Y212 and W235 residues that make cation-π contacts with the trimethylammonium group. Both aromatic residues are essential in the H3K4me3 recognition, as substitution of these residues with Ala disrupts the interaction. Unlike the wild-type ING1, the W235A mutant, overexpressed in the stable clones of melanoma cells or in HT1080 cells, was unable to stimulate DNA repair after UV irradiation or promote DNA-damage-induced apoptosis, indicating that H3K4me3 binding is necessary for these biological functions of ING1. Furthermore, N216S, V218I, and G221V mutations, found in human malignances, impair the ability of ING1 to associate with H3K4me3 or to induce nucleotide repair and cell death, linking the tumorigenic activity of ING1 with epigenetic regulation. Together, our findings reveal the critical role of the H3K4me3 interaction in mediating cellular responses to genotoxic stresses and offer new insight into the molecular mechanism underlying the tumor suppressive activity of ING1.     

Differential contribution of basic residues to HIV-1 nucleocapsid protein’s nucleic acid chaperone function and retroviral replication

The human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) protein contains 15 basic residues located throughout its 55-amino acid sequence, as well as one aromatic residue in each of its two CCHC-type zinc finger motifs. NC facilitates nucleic acid (NA) rearrangements via its chaperone activity, but the structural basis for this activity and its consequences in vivo are not completely understood. Here, we investigate the role played by basic residues in the N-terminal domain, the N-terminal zinc finger and the linker region between the two zinc fingers. We use in vitro ensemble and single-molecule DNA stretching experiments to measure the characteristics of wild-type and mutant HIV-1 NC proteins, and correlate these results with cell-based HIV-1 replication assays. All of the cationic residue mutations lead to NA interaction defects, as well as reduced HIV-1 infectivity, and these effects are most pronounced on neutralizing all five N-terminal cationic residues. HIV-1 infectivity in cells is correlated most strongly with NC’s NA annealing capabilities as well as its ability to intercalate the DNA duplex. Although NC’s aromatic residues participate directly in DNA intercalation, our findings suggest that specific basic residues enhance these interactions, resulting in optimal NA chaperone activity.     

Sequestosome 1/p62 links familial ALS mutant SOD1 to LC3 via an ubiquitin-independent mechanism

The p62/sequestosome 1 protein has been identified as a component of pathological protein inclusions in neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). P62 has also been implicated in autophagy, a process of mass degradation of intracellular proteins and organelles. Autophagy is a critical pathway for degrading misfolded and/or damaged proteins, including the copper-zinc superoxide dismutase (SOD1) mutants linked to familial ALS. We previously reported that p62 interacted with ALS mutants of SOD1 and that the ubiquitin-association domain of p62 was dispensable for the interaction. In this study, we identified two distinct regions of p62 that were essential to its binding to mutant SOD1: the N-terminal Phox and Bem1 (PB1) domain (residues 1–104) and a separate internal region (residues 178–224) termed here as SOD1 mutant interaction region (SMIR). The PB1 domain is required for appropriate oligomeric status of p62 and the SMIR is the actual region interacting with mutant SOD1. Within the SMIR, the conserved W184, H190 and positively charged R183, R186, K187, and K189 residues are critical to the p62–mutant SOD1 interaction as substitution of these residues with alanine resulted in significantly abolished binding. In addition, SMIR and the p62 sequence responsible for the interaction with LC3, a protein essential for autophagy activation, are independent of each other. In cells lacking p62, the existence of mutant SOD1 in acidic autolysosomes decreased, suggesting that p62 can function as an adaptor between mutant SOD1 and the autophagy machinery. This study provides a novel molecular mechanism by which mutant SOD1 can be recognized by p62 in an ubiquitin-independent fashion and targeted for the autophagy–lysosome degradation pathway.