Trimeresurus stejnegeri snake venom plasminogen activator - Site-directed mutagenesis and molecular modeling

The specific plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) is a serine proteinase presenting 23% sequence identity with the proteinase domain of tissue type plasminogen activator, and 63% with batroxobin, a fibrinogen clotting enzyme from Bothrops atrox venom that does not activate plasminogen. TSV-PA contains six disulfide bonds and has been successfully overexpressed in Escherichia coli (Zhang, Y., Wisner, A., Xiong, Y. L,, and Bon, C, (1995) J. Biol. Chem. 270, 10246-10255), To identify the functional domains of TSV-PA, we focused on three short peptide fragments of TSV-PA showing important sequence differences with batroxobin and other venom serine proteinases. Molecular modeling shows that these sequences are located in surface loop regions, one of which is next to the catalytic site, When these sequences were replaced in TSV-PA by the equivalent batroxobin residues none generated either fibrinogen-clotting or direct fibrinogenolytic activity, Two of the replacements had little effect in general and are not critical to the specificity of TSV-PA for plasminogen. Nevertheless, the third replacement, produced by the conversion of the sequence DDE 96a-98 to NVI, significantly increased the K-m for some tripeptide chromogenic substrates and resulted in undetectable plasminogen activation, indicating the key role that the sequence plays in substrate recognition by the enzyme.

RecA-mediated, targeted mutagenesis in zebrafish

We have evaluated the efficacy of RecA, a prokaryotic protein involved with homologous recombination, to direct site-specific mutagenesis in zebrafish embryos. For this we coinjected a vector containing a mutated enhanced green fluorescent protein (EGFP) gene plus 236-nucleotide corrective single-stranded DNAs coated with RecA into I-cell zebrafish embryos. Twenty-hours after fertilization, about 5% to 20% of injected embryos showed EGFP expression in I or more cells when RecA-coated corrective DNAs were used, but not when RecA was omitted. Mutated EGFP genes with 1-bp insertions or deletions were inefficiently activated, whereas those with 7-bp insertions were activated about 4-fold more efficiently. RecA-coated template strand had a higher efficiency than its complementary strand in activation of EGFP expression. Prior irradiation of the embryos with UV light enhanced RecA-mediated restoration of gene activity, suggesting that the effects we observed were augmented by one or more factors of zebrafish DNA repair systems.

80 MeV/u C-12离子诱变选育PHB高产菌株；A strain of high PHB output by mutagenesis of 80 MeV/u C-12 ions

用80MeV/u12C6+离子对一株积累聚β-羟基丁酸酯(PHB)的芽孢杆菌(BacillusP-9)进行诱变选育,最终获得PHB高产株菌G15,其PHB产量达2.93g/L,与出发菌株相比较,PHB积累量提高了1.5倍。研究结果表明,本试验辐照最佳剂量为15Gy。重离子辐照诱变育种效果显著,具有广阔的发展应用前景。

低能N+诱导拟南芥突变体DNA变异的研究

低能离子束的诱变效应首先由我国科学家发现并将其广泛应用于育种实践，但是离子注入诱导DNA变异的研究结果主要是以微生物离体质粒DNA为材料获得的，以活体高等生物为材料的研究尚未见报道。 我们以30 keV N+（注入剂量80×1015 ions/cm2）注入拟南芥后获得的稳定突变体T80II为实验材料，对突变体植株进行了RAPD标记，并将T80II和对照部分RAPD特异条带进行克隆测序和DNA序列分析。结果显示，在可分辨的总计397个RAPD条带中，T80II株系中有52个条带表现出差异，包括条带的缺失和增加，条带变异率为13.1%;克隆的T80II序列中，平均每16.8个碱基出现一个碱基变异位点，表现出较高频率的碱基突变。碱基突变的类型包括碱基的颠换、转换、缺失、插入等。在检测到的275个碱基突变中，主要是单碱基置换（97.09%），碱基缺失或者插入的比例较小（2.91%）。在碱基置换中，转换的频率（66.55%）高于颠换的频率（(30.55%)。此外，构成DNA的四种碱基均可以被离子束辐照诱发变异，而且每一种碱基都可以被其它三种碱基所替换，但是胸腺嘧啶（T）的辐射敏感性要高于其它三种碱基。通过分析突变碱基周边序列，对低能N+离子注入拟南芥突变体引发的碱基突变热点进行了讨论。 另外，低能离子注入诱变获得的突变体特异表达基因的克隆方面也没有报道。我们以突变体T80II作为实验材料，用PCR增效的减法杂交技术构建了T80II特异表达的cDNA减法文库，克隆特异表达的cDNA片段，并对其中1个与14-3-3 protein GF14 nu (GRF7) gene有部分同源性、长712 bp的cDNA片段进行了讨论。我们的研究证明通过减法杂交技术克隆低能离子诱发的突变体特异表达的cDNA是可能的，这为低能离子注入技术在分子生物学上的应用开辟了一个新思路。

Cyt b-559 链Arg18和Ser24的定点突变及其对PSII功能的影响

Cyt b-559是光系统II反应中心的成分之一，它由亚基和亚基组成的。在Cyt b-559中，血红素辅基与两个亚基中的组氨酸连接成有功能的蛋白，并维持PSII的功能稳定性。前人曾将与血红素相连的His突变，导致Cyt b-559功能和PSII稳定性的丧失。基于此研究，本文采用定点突变技术，将亚基中与His23位置最近的上游氨基酸Arg18分别用Gly和Glu取代，下游氨基酸Ser24用Phe取代，获得了衣藻Cyt b-559的突变体。对突变体的分析，有以下新结果：突变体都能进行光合自养，但无论在异养培养基上还是自养培养基上，和对照相比，其生长速度非常缓慢; PSII的活性分析，表明PSII的放氧活性为野生衣藻细胞的50%～80%， Fv/Fm 的荧光参数为40%～70%;对突变体进行强光（1000μE•m-2•s-1）照射，10min后，其放氧活性都降低为0，而野生型衣藻还保持35%的活性;提取类囊体膜蛋白，进行SDS-PAGE电泳和Western-blotting分析，显示突变体的膜蛋白与对照无显著差异。这些结果说明对围绕血红素环境的固有氨基酸的改变，虽然并没有明显影响类囊体膜蛋白的表达和组成，但是却影响了衣藻细胞的生长和PSII的活性，增加了衣藻细胞对强光的敏感性，降低了衣藻细胞自身的光保护能力。这说明靠近血红素配位环境的氨基酸Arg和Ser，尤其是Arg，对Cyt b-559的功能维持不可缺少，对于维持PSII的活性也很重要。

光系统II大量捕光色素蛋白复合体（LHCIIb）结构和功能关系 的定点突变研究

定点突变技术可以对某个已知基因的特定碱基进行定点改变、缺失或者插入，从而改变对应的氨基酸序列和蛋白质结构，因而成为研究蛋白质结构和功能之间的复杂关系的有力工具。对突变基因的表达产物进行研究有助于我们了解蛋白质结构和功能的关系，探讨蛋白质的结构/结构域。 植物体光系统II的大量捕光色素蛋白复合体（LHCIIb）具有多种功能，在自然界不同日光光强下分别执行捕获、传递光能或将过度激发能非光化学耗散的功能。最新的近原子分辨率LHCIIb晶体结构揭示出在LHCIIb穿膜螺旋B/C之间的环区具有复杂的超二级结构，其中一个新发现就是在此环区靠近穿膜螺旋C的区域中存在一个反平行股的结构，其功能不明。为了研究此反平行链对于LHCIIb复合体在结构和功能上的意义，我们将了这一区域的3个氨基酸（Val119、His120、Ser123）分别定点突变成Phe、Leu和Gly，并研究了这三个定点突变对LHCIIb结构和功能上的影响。结果如下：1，CD光谱揭示出该反平行链对于调节新黄质及其附近色素群的构象十分重要。虽然这三个突变只造成很少的新黄质丢失（V119F, 0.09; S123G, 0.17; and H120L, 0.26），但是却使色素构象发生了巨大变化。2，将S123突变成G导致复合物对光破坏更加敏感并且更易于聚集，在介质酸化后复合物的荧光淬灭更加显著。这些结果说明这段反平行链对于调节LHCIIb色素构象以及控制LHCIIb聚集体形成和叶绿素荧光产量具有重要作用。 以结构为基础的计算设计方法与定向进化相结合是蛋白质工程的一个发展方向。最近，通过计算设计已成功地向蛋白质引入了新的催化活性、提高了蛋白质的稳定性、设计了酶的催化活性位点、改变了酶的底物特异性等. 目前还没有见到有研究定向地，以理性方式对LHCIIb进行蛋白质设计。我们使用蛋白质的计算机辅助设计工具——RosettaDesign鉴定出一个可以显著提高LHCIIb光、热稳定性的定点突变I124L，并且突变体的的结构和功能与野生型无异。这是首次将计算机辅助设计应用于提高LHCIIb稳定性的研究。

Strikingly different penetrance of LHON in two Chinese families with primary mutation G11778A is independent of mtDNA haplogroup background and secondary mutation G13708A

The penetrance of Leber's hereditary optic neuropathy (LHON) in families with primary mitochondrial DNA (mtDNA) mutations is very complex. Matrilineal and nuclear genetic background, as well as environmental factors, have been reported to be involved in d

Anti-HIV-1 property of trichosanthin correlates with its ribosome inactivating activity

Trichosanthin (TCS) is a type I ribosome inactivating (RI) protein possessing anti-tumor and antiviral activity, including human immunodeficiency virus (HIV). The mechanism of these actions is not entirely clear, but is generally attributed to its RI property. In order to study the relationship between the anti-HIV-1 activity of TCS and its RI activity, three TCS mutants with different RI activities were constructed by using site-directed mutagenesis. The anti-HIV-1 activities of the three mutants were tested in vitro. Results showed that two TCS mutants, namely TCSM((120-123)), TCSE160A/E189A, with the greatest decrease in RI activity, lost almost all of the anti-HIV activity and cytopathic effect. Another mutant TCSR122G, which exhibited a 160-fold decrease in RI activity, retained some anti-HIV activity. The results from this study suggested that RI activity of TCS may have significant contribution to its anti-HIV-1 property. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Independency of anti-HIV-1 activity from ribosome-inactivating activity of trichosanthin

Trichosanthin (TCS) is a type I ribosome-inactivating (RI) protein possessing multiple biological and pharmacological activities. Its major action is inhibition of human immunodeficiency virus (HIV) replication but the mechanism is still elusive. All evidences showed that this action is related to its RI activity. Previous studies found that TCS mutants with reduced RI activity simultaneously lost some anti-HIV activity. In this study, an exception was demonstrated by two TCS mutants retaining almost all RI activity but were devoid of anti-HIV-1 activity. Five mutants were constructed by using site-directed mutagenesis with either deletion or addition of amino acids to the C-terminal sequence. Results showed that the RI activity of mutants with C-terminal deletion mutants (TCSC2, TCSC4, and TCSC14) decreased by 1.2-3.3-fold with parallel downshifting of its anti-HIV-1 activity (1.4-4.8-fold). Another two mutants, TCSC19aa and TCSKDEL having 19 amino acid extension and a KDEL signal sequence added to the C-terminal sequence, retained all RI activity but subsequently lost most of the anti-HIV-1 activity. These findings suggested that ribosome inactivation alone might not be adequate to explain the anti-HIV action of TCS. (C) 2003 Elsevier Science (USA). All rights reserved.

Site-directed PEGylation of trichosanthin retained its anti-HIV activity with reduced potency in vitro

Trichosanthin (TCS) was the first ribosome inactivating protein found to possess anti-HIV-1 activity. Phase I/II clinical trial of this compound had been done. Antigenicity and short plasma half-life were the major side effects preventing further clinical trial. Modification of TCS is therefore necessary to revive the interest to develop this compound as an anti-HIV agent. Three potential antigenic sites (Ser-7, Lys-173, and Gln-219) were identified by computer modeling. Through site-directed mutagenesis, these three antigenic amino acids were mutated to a cysteine residue resulting in 3 TCS mutants, namely S7C, K173C, and Q219C. These mutants were further coupled to polyethylene glycol with a molecular size of 20 kDa (PEG) via the cysteine residue. This produced another three TCS derivatives, namely PEG(20)k-S7C, PEG(20)k-K173C, and PEG(20)k-Q219C. PEGylation had been widely used recently to decrease immunogenicity by masking the antigenic sites and prolong plasma half-life by expanding the molecular size. The in vitro anti-HIV-1 activity of these mutants and derivatives was tested. Results showed that the anti-HIV-1 activity of S7C, K173C, and Q219C was decreased by about 1.5- to 5.5-fold with slightly lower cytotoxicity. On the other hand, PEGylation produced larger decrease (20- to 30-fold) in anti-HIV activity. Cytotoxicity was, however, weakened only slightly by about 3-fold. The in vitro study showed that the anti-HIV activity of PEGylated TCS was retained with reduced potency. The in vivo activity is expected to have only slightly changed due to other beneficial effects like prolonged half-life. (C) 2004 Elsevier Inc. All rights reserved.

Characterize dynamic conformational space of human CCR5 extracellular domain by molecular modeling and molecular dynamics simulation

The chemokine receptor CCR5 is the receptor for several chemokines and major coreceptor for R5 human immunodeficiency virus type-1 strains entry into cell. Three-dimensional models of CCR5 were built by using homology modeling approach and 1 ns molecular dynamics (MD) simulation, because studies of site-directed mutagenesis and chimeric receptors have indicated that the N-terminus (Nt) and extracellular loops (ECLs) of CCR5 are important for ligands binding and viral fusion and entry, special attention was focused on disulfide bond function, conformational flexibility, hydrogen bonding, electrostatic interactions, and solvent-accessible surface area of Nt and ECLs of this protein part. We found that the extracellular segments of CCR5 formed a well-packet globular domain with complex interactions occurred between them in a majority of time of MID simulation, but Nt region could protrude from this domain sometimes. The disulfide bond Cys20-Cys269 is essential in controlling specific orientation of Nt region and maintaining conformational integrity of extracellular domain. RMS comparison analysis between conformers revealed the ECL1 of CCR5 stays relative rigid, whereas the ECL2 and Nt are rather flexible. Solvent-accessible surface area calculations indicated that the charged residues within Nt and ECL2 are often exposed to solvent. Integrating these results with available experimental data, a two-step gp120-CCR5 binding mechanism was proposed. The dynamic interaction of CCR5 extracellular domain with gp120 was emphasized. (C) 2004 Elsevier B.V. All rights reserved.

Distinct Evolutionary Patterns Between Two Duplicated Color Vision Genes Within Cyprinid Fishes

We investigated the molecular evolution of duplicated color vision genes (LWS-1 and SWS2) within cyprinid fish, focusing on the most cavefish-rich genus-Sinocyclocheilus. Maximum likelihood-based codon substitution approaches were used to analyze the evolution of vision genes. We found that the duplicated color vision genes had unequal evolutionary rates, which may lead to a related function divergence. Divergence of LWS-1 was strongly influenced by positive selection causing an accelerated rate of substitution in the proportion of pocket-forming residues. The SWS2 pigment experienced divergent selection between lineages, and no positively selected site was found. A duplicate copy of LWS-1 of some cyprinine species had become a pseudogene, but all SWS2 sequences remained intact in the regions examined in the cyprinid fishes examined in this study. The pseudogenization events did not occur randomly in the two copies of LWS-1 within Sinocyclocheilus species. Some cave species of Sinocyclocheilus with numerous morphological specializations that seem to be highly adapted for caves, retain both intact copies of color vision genes in their genome. We found some novel amino acid substitutions at key sites, which might represent interesting target sites for future mutagenesis experiments. Our data add to the increasing evidence that duplicate genes experience lower selective constraints and in some cases positive selection following gene duplication. Some of these observations are unexpected and may provide insights into the effect of caves on the evolution of color vision genes in fishes.

U19/Eaf2 Binds to and Stabilizes von Hippel-Lindau Protein

Studies have firmly established a key regulatory role for the tumor suppressor pVHL in the regulation of the vascular system and normal spermatogenesis. Here, we report that knockout of the newly identified tumor suppressor U19/Eaf2 also caused vascular system abnormalities and aspermatogenesis, suggesting a potential link between U19/Eaf2 and pVHL. Coimmunoprecipitation and in vitro binding assays showed an association between U19/Eaf2 and pVHL, whereas deletion mutagenesis revealed the requirement of the NH2 terminus of U19/Eaf2 and both the alpha and beta domains of pVHL for this binding. U19/Eaf2 stabilizes pVHL, as shown by protein stability and pulse-chase studies. Testes and mouse embryonic fibroblasts (MEF) derived from U19/Eaf2 knockout mice expressed reduced levels of pVHL, indicating that full in vivo expression of pVHL indeed requires U19/Eaf2. As expected, U19/Eaf2 knockout MEF cells exhibited an increased level and activity of hypoxia-inducible factor 1 alpha (HIF1 alpha), a protein typically regulated via a pVHL-mediated degradation pathway. Furthermore, angiogenesis in a Matrigel plug assay was significantly increased in U19/Eaf2 knockout mice. The above observations argue that U19/Eaf2 can modulate HIF1 alpha and angiogenesis, possibly via direct binding and stabilization of pVHL. [Cancer Res 2009;69(6):2599-606]