Kunitz抑制剂家族成员抑肽酶对纤溶酶原活化的抑制作用

抑肽酶属Kunitz抑制剂家族成员，能够抑制激肽释放酶、纤溶酶及胰蛋白酶的蛋白水解活性．研究表明，抑肽酶能够抑制尿激酶型-纤溶酶原激活刹（u—PA）和组织型纤溶酶原激活剂（t—PA）对纤溶酶原的激活，但不影响u—PA和t—PA对小分子底物的酰胺水解活性．用u—PA研究了上述作用的机制，发现抑肽酶与u—PA的丝氨酸蛋白酶功能区特异性结合，而与纤溶酶原没有相互作用．抑肽酶与uPA的结合并不阻断u—PA的活性位点，因为u—PA对小分子底物的水解活性仍然保持．上述发现提示抑肽酶可能存在另一种抑制作用模式，该模式不同于以前报道的关于Kunitz抑制剂或纤溶酶原激活酶抑制剂的作用．由于人体内的Kunitz抑制剂与抑肽酶在结构上非常相似，根据研究结果，推测体内纤溶酶原的激活作用并非仅受丝氨酸蛋白酶抑制剂的控制。     

CCR5膜外二硫键Cys20-Cys269对其构象完整性以及功能行使起关键作用

人类化学趋化因子受体CCR5是HIV-1病毒进入人体细胞的主要辅助受体。实验表明,CCR5分子的N末端区域对化学趋化因子结合以及HIV病毒入侵起关键作用。用同源模建的方法构建CCR5结构模型,并对该模型的胞外结构域进行了1ns高温分子动力学和200ps常温分子动力学模拟。结果表明,CCR5的胞外结构域整体上表现为一个包装紧密的球形构象,二硫键Cys20-Cys269对这一构象的形成以及N末端区域的空间取向起关键作用,同时,二硫键的存在使N末端1-19区域定位在胞外结构域的顶部,并增加N末端构象柔性,使其有机会伸展到胞外空间去。根据这一结果和现有的实验证据,提出了HIV-CCR5两步结合机制。     

人尿胰酶抑制剂部分生化性质

用自制人尿胰酶抑制剂（HUTI）,研究了它的部分生物化学性质。经HPLC和凝胶排阻层析表明,纯HUTI的表观分子量与牛血清白蛋白相当,约为66kD。然而,10％SDS－PAGE分析表明,它的分子量约为40kD,这一差异可能与蛋白分子具有高含量 的糖链有关。另外,对HUTI的氨基酸组成、糖含量和抑制类型等亦进行了研究。     

Structural basis of latency in plasminogen activator inhibitor-1.

Human plasminogen activator inhibitor-1 (PAI-1) is the fast-acting inhibitor of tissue plasminogen activator and urokinase and is a member of the serpin family of protease inhibitors. Serpins normally form complexes with their target proteases that dissociate very slowly as cleaved species and then fold into a highly stable inactive state in which the residues that flank the scissile bond (P1 and P1';) are separated by about 70 A. PAI-1 also spontaneously folds into a stable inactive state without cleavage; this state is termed 'latent' because inhibitory activity can be restored through denaturation and renaturation. Here we report the structure of intact latent PAI-1 determined by single-crystal X-ray diffraction to 2.6 A resolution. The three-dimensional structure reveals that residues on the N-terminal side of the primary recognition site are inserted as a central strand of the largest beta sheet, in positions similar to the corresponding residues in the cleaved form of the serpin alpha 1-proteinase inhibitor (alpha 1-PI). Residues C-terminal to the recognition site occupy positions on the surface of the molecule distinct from those of the corresponding residues in cleaved serpins or in the intact inactive serpin homologue, ovalbumin, and its cleavage product, plakalbumin. The structure of latent PAI-1 is similar to one formed after cleavage in other serpins, and the stability of both latent PAI-1 and cleaved serpins may be derived from the same structural features.     

内生枯草芽孢杆菌HD-1β-甘露聚糖酶基因的克隆及结构生物学分析

采用PCR技术从一株内生枯草芽孢杆菌HD-1基因组DNA中扩增出β-甘露聚糖酶基因核苷酸编码序列.序列分析表明,该基因全长1 089bp,编码362个氨基酸和一个终止密码子,N端前27个氨基酸为其信号肽.该酶氨基酸序列与相同来源的β-甘露糖苷酶同源性最高达98.34%,具有ManB保守结构域,属于糖苷水解酶家族26的一员.通过对该酶分子三维结构的预测分析,该酶的催化域形成TIM桶状结构,Cys92和Cys112形成二硫键,它们之间的部分构成该酶的氧化还原反应的活性中心,Glu100和Glu292分别为该酶的酸碱催化位点和亲核催化位点.三维结构的分析为提高酶的催化活性的和功能方面的定向改造提供了依据.     

Peptide exosite inhibitors of factor VIIa as anticoagulants

Potent anticoagulants have been derived by targeting the tissue factor-factor VIIa complex with naive peptide libraries displayed on M13 phage. The peptides specifically block the activation of factor X with a median inhibitory concentration of 1 nM and selectively inhibit tissue-factor-dependent clotting. The peptides do not bind to the active site of factor VIIa; rather, they work by binding to an exosite on the factor VIIa protease domain, and non-competitively inhibit activation of factor X and amidolytic activity. One such peptide (E-76) has a well defined structure in solution determined by NMR spectroscopy that is similar to the X-ray crystal structure when complexed with factor VIIa. These structural and functional studies indicate an allosteric 'switch' mechanism of inhibition involving an activation loop of factor VIIa and represent a new framework for developing inhibitors of serine proteases.     

Inducible expression pattern of rice Bowman-Birk inhibitor gene<Emphasis Type="Italic">OsWIP1-2</Emphasis> and its protease inhibitory activity

The WIP1-2 gene was cloned from rice. It be-longs to the Bowman-Birk inhibitor gene family. Northernblot showed that expression of this gene was induced bywounding and jasmonic acid (JA). It indicates that the OsWIPI gene plays an important role in the rice defense sys-tem. The OsWIP1-2 was cloned into pET28a and expressed inE. coli. Its expressed product was purified in the form offusion protein and tested for the inhibitory activities againsttrypsin and chymotrypsin. It was found that the fusion pro-tein could inhibit chymotrypsin, but not trypsin. It was alsofound that the His tag at its C-terminal affected its inhibitoryactivity significantly. The fusion protein with a naturalC-terminal had the inhibitory activity, while no inhibitoryactivity was detected in the fusion protein with a (His)6-tag atits C-terminal. This implies that extra amino acid residues atthe C-terminal of OsWIP1-2 may interfere with its correctfolding. The inhibitory assay indicated that the members ofrice Bowman-Birk inhibitor gene family probably differenti-ated both in their structure and function.     

Dithiothreitol decreases the thermal stability and unfolding cooperativity of ribulose-1, 5-bisphosphate carboxylase/oxygenase

Plant rubisco consists of eight large subunits (55 kD) encoded by chloroplast gene and eight small subunits (15 kD) encoded by nuclear gene. There are abundant cysteine residues that do not form disulfide bonds in native rubisco. Differential scanning calorimetry has been used to study some plant rubisco and suggested an irreversible two-state denaturation due to the high cooperativity in subunits. By comparing the data from circular dichroism, fluorescence, differential scanning calorimetry, SDS electrophoresis, and activity assays in the absence or presence of DTT, we suggest that the formation of disulfide bonds in subunits during the early thermal unfolding may increase the thermal stability and the thermal unfolding cooperativity of rubisco.     

人Elp3在毕赤酵母中的分泌表达及HAT活性测定

人Elp3蛋白(human Elongator protein 3,hElp3)是组蛋白乙酰转移酶(Histone acetyltranferase,HAT)Elongator复合物的催化亚基,可参与组蛋白乙酰化修饰与基因转录延伸,其功能异常与人类多种疾病相关.目前对其羧基末端高度保守的第二功能域研究较少.以pYES2hElp3质粒为模板,PCR获hElp3基因全长,插入改造的毕赤酵母表达载体pPIC9KH,转化巴斯德毕赤酵母菌GS115菌株.经表型鉴定、PCR分析和G418筛选得到Mut+型多拷贝整合菌.0.5%的甲醇诱导hElp3高效分泌表达,Ni-NTA纯化及Western印迹证实获目的蛋白.OPA法测得其拥有良好的HAT活性,为体外测定其第二结构域是否拥有催化活性奠定了基础,对筛选抑制其HAT活性的小分子药物用于疾病治疗研究至关重要.     

小麦高分子量麦谷蛋白亚基1By15N端区同源建模与分子动力学模拟

通过二级结构预测、折叠识别、同源建模和分子动力学模拟的方法,获得三个小麦高分子量麦谷蛋白亚基1By15 N端区的三维结构模型.模型分析发现,y 型高分子量麦谷蛋白亚基N端的5个半胱氨酸具有两种成键模式,一种是Cys22和Cys44之间形成一个链内二硫键,N端剩余的三个半胱氨酸Cys10,45和55都是自由半胱氨酸用于形成链间二硫键;另一种是Cys22和Cys44,Cys10和Cys55之间形成两对链内二硫键,剩余一个Cys45作为自由半胱氨酸用于形成链间二硫键.     

酵母Elp3在毕赤酵母中的分泌表达及活性测定

酵母Elp3是Elongator复合物的催化亚基,可参与组蛋白乙酰化修饰与基因转录延伸.生物信息学分析及有关研究表明Elp3除组蛋白乙酰转移酶(Histone acetyltranferase,HAT)活性外,可能还拥有组蛋白去甲基化酶活性.本文以yElp3的pYES2yElp3质粒为模板,PCR获得yElp3基因全长,插入改造过的毕赤酵母表达载体pPIC9KH,转化巴斯德毕赤酵母GS115菌株.经表型鉴定、PCR分析及G418筛选获Mut+型多拷贝整合菌,经0.5%的甲醇诱导和Ni-NTA纯化,得到目的蛋白并对其进行体外酶活测定.SDS-PAGE分析表明yElp3在毕赤酵母中实现了高效分泌表达,Western印迹证实得到的蛋白为yElp3.OPA法测得纯化蛋白拥有较好的HAT活性,具有生物活性的Elp3蛋白的获得为体外测定其第二结构域是否有催化活性奠定了基础.     

基于喹喔啉酮结构的醛糖还原酶抑制剂的设计合成与构效关系研究

本文以喹喔啉酮为母核结构,在其N1位引入羧基,同时对C3位侧链结构进行优化,设计合成了一系列喹喔啉酮衍生物作为醛糖还原酶抑制剂候选物.活性检测结果表明,所有化合物均显示出明显的醛糖还原酶抑制活性.其中,2-（3-（4-羟基苯丙烯基）-2-氧喹喔啉-1（2H）-烷基）乙酸（ 4d ）展现出最强的抑制活性,IC₅₀值为93 nmol/L,与目前上市的epalrestat活性相当（IC₅₀=83 nmol/L）.另外,分子对接研究表明化合物 4d 与醛糖还原酶的活性位点结合比较紧密.      

野花生豆凝集素半胱氨酸残基的研究

将野花生豆经浸取、硫酸铵分级沉淀、猪胃粘蛋白-Sepharose4B亲和层析和SephacrylS-200HR分子筛层析后,可得到对人类A型血专一凝集的野花生豆凝集素（Crotalaria mucronata Lectin,CML),纯化的CML在PAGE和SDS-PAGE中均显示单一蛋白带,在含8mol/L,脲和不含脲的缓冲体系中,分别用NEM修饰CML中的半胱氨酸残基,经计算被修饰巯基数目分别为3.1个和3.2个,且修饰后的CML的活性仍不丧失,通过NR/R单向SDS-PAGE和NR/R双向对角线SDS-PAGE研究,发现CML不含二硫键,表明CML中的半胱氨酸残基既与CML的凝集活性无关,也不形成稳定蛋白质结构的二硫键。     

Purification and Partial Characterization of a Collagenolytic Enzyme Produced by Pseudomonas aeraginosa SCU Screened from Rotten Hides

Strain Pseudomonas Aeraginosa SCU isolated from rotten hides is shown to produce various gelatinolytic enzymes with molecular masses ranging from - 50 to - 200 kD. A gelatinolytic enzyme called PAC exhibiting collagenolytic activity is purified by SP sepharose fast flow, Sephadex G-200 gel filtration and native PAGE cutting method. The purified enzyme has an apparent molecular weight of about 110 kD by SDS PAGE without β-mercaptoethanol. Treatment withtβ-Me suggests that PAC is dissociated into three subunits approximately 33 kD, 25 kD and 20 kD with a ratio of 2:1:1, named sub A, sub B and sub C repectively. EDFA and EGTA display a significant inhibitory effect on the enzyme activity while PMSF, leupeptin and pepstain do not appreciably inhibit it. The first 15 amino acid residues of the major subunit (subA) are determined and the sequence is Ala-Glu-Ala-Gly-Gly-Pro-Gly-Gly-Asn-Gln-Lys-Ile-Gly -Lys-Tyr. This sequence is identical to that of elastase of P. aeruginosa. The fragment of encoding mature sub A is cloned and its sequence is determined, which has a high homology with the gene of elastase. These results indicate that PAC is a novel collagenolytic metalloprotease composed of three kinds of subunits, of which elastase is the major one.     

蜂毒肽类似物的基因表达与构效关系研究

根据先前建立的蜂毒肽QSAR模型预测，为获得溶血活性相对最低，保留或提高抑菌活性的蜂毒肽类似物。设计了六条蜂毒肽类似物基因，采用P. pastoris表达系统，通过构建 pPICZα-A-Mel重组质粒，电击转化酵母。以α-factor为分泌信号，在GS115中分泌表达。以MDH、MMH筛选，诱导培养，6×His亲和层析纯化表达产物, Tricine-SDS-PAGE电泳证实。新设计的六条类似物基因表达后抑菌活性与天然序列蜂毒肽相比，四条增强，两条减弱。类似物【C】抑菌效价(U)为1.29，是重组天然序列蜂毒肽的1.6倍左右，活性最强，而重组天然序列蜂毒肽为0.7977。类似物【C】溶血活性比天然序列蜂毒肽降低至1/25。类似物【B】的表达量最高，为0.31mg/mL，其抑菌活性仅次于【C】。两种类似物的溶血活性降低至天然序列蜂毒肽的1/25左右，两种降低至1/20左右，还有两种降低至1/15左右。对蜂毒肽分子结构的优化设计合理，达到了保留或提高抑菌活性，降低溶血活性的目的。     

珙桐种子层积过程中抑制物活性的变化

为探明珙桐种子不同部位内源抑制物含量及其在层积过程中的变化情况,分别取层积12、16个月的珙桐种子,以甲醇和丙酮为提取剂,制备其种仁(胚和胚乳)、种壳的浸提液,分析各浸提液对白菜籽发芽的影响。结果表明:珙桐种仁和种壳中均含有发芽抑制物质; 随着浸提液质量浓度的升高,抑制物生理活性增强; 种仁浸提液抑制物的生理活性显著高于种壳浸提液; 种仁甲醇浸提液抑制物的生理活性显著高于丙酮浸提液,而两种提取剂中,种壳浸提液的生理活性差异不显著。低温层积12个月再变温层积4个月后,珙桐种子内源抑制物的生理活性显著降低,但此时种仁 0.09 g/mL的甲醇浸提液对白菜籽的发芽指数仍有显著的抑制作用,表明此时种仁中仍含有一定量的抑制物质。     

Functional significance of the Kunitz-type inhibitory domains of lipoprotein-associated coagulation inhibitor

Blood coagulation can be initiated when factor VII or VIIa, a plasma protease, binds to its essential cofactor, tissue factor (TF), and proteolytically activates factors IX and X, triggering a cascade of events which eventually leads to the formation of thrombin and a fibrin clot. Plasma contains a lipoprotein-associated coagulation inhibitor (LACI) which inhibits activated factor X (Xa) directly and, in a Xa-dependent way, inhibits VII(a)/TF activity, presumably by forming a quaternary Xa/LACI/VII(a)/TF complex. Sequence analysis of complementary DNA clones has shown that LACI contains three tandemly repeated Kunitz-type serine protease inhibitory domains. To investigate the relationship between these Kunitz structures and LACI function, we have used site-directed mutagenesis to produce altered forms of LACI in which the residue at the active-site cleft of each Kunitz domain has been individually changed. The second Kunitz domain is required for efficient binding and inhibition of Xa, and both Kunitz domains 1 and 2 are required for the inhibition of VIIa/TF activity; but alteration of the active-site residue of the third Kunitz domain has no significant effect on either function. We propose that in the putative inhibitory complex, Kunitz domain 1 is bound to the active site of VII(a)/TF and that Kunitz domain 2 is bound to Xa's active site.     

水稻土壤宏基因组中的β-葡萄糖苷酶基因克隆、表达及酶活的研究

β-葡萄糖苷酶(Ec3.2.1.21)属于糖苷水解酶家族3,它能够水解非还原性末端的β-D葡萄糖苷键,释放出游离的葡萄糖及相应的配基。β-葡萄糖苷酶是纤维素降解中的关键酶,对于可再生资源纤维素的利用具有十分重要的意义。本研究从水稻土壤中分离得到β-葡萄糖苷酶基因pds5,将其克隆到表达载体pET32a(+)中,转化BL21大肠杆菌中,并诱导表达该基因。重组BL21大肠杆菌用IPTG诱导后,所提取的酶蛋白具有β-葡萄糖苷酶的活性,经SDS-PAGE分析,确定其相对分子质量为83 kD。通过控制pH和温度的方法,测得该酶酶活最适pH为7.0,最适温度为37.5℃。     

新型磺酰类醛糖还原酶抑制剂的合成及活性研究

以2,4-二甲苯胺为原料,设计并合成了一系列环外磺酰胺(酯)类衍生物,对目标化合物的醛糖还原酶(ALR2)抑制活性进行了测定,并利用分子对接方法研究了抑制剂与醛糖还原酶蛋白的结合模式. 结果显示环外磺酰类化合物8a-d, 12a-b, 16a-b具有良好的体外醛糖还原酶抑制活性,分子模拟预测其能与酶蛋白的活性位点空腔较好结合. 化合物8a-d是一类新型醛糖还原酶抑制剂,活性良好,并可作为先导化合物探索活性更高的醛糖还原酶抑制剂.