Mg2+和Na+对多核酶系统体外切割反应的影响

为了进一步了解2价Mg2+和1价Na+存在与否的情况下,多核酶系统对底物RNA的切割效率,构建了pGEM-Coat'A,pGEM-Coat'A196Rz质粒和pGEM-MDR1靶质粒,通过用SP6/T7转录试剂盒在体外转录RNA,在无细胞系统进行切割反应,反应产物通过6%变性聚丙烯酰胺凝胶电泳,干胶、x光片曝光自显影,利用Image J生物图像分析软件分析,结果表明,多核酶系统的切割效率依赖于二价Mg2+的浓度,切割产物随Mg2+浓度的增加而增加,而且具有反应时间的依赖性,在Na+浓度低于200 mmol/L且单独存在时,没有切割产物生成,相反,在Na+和Mg2+共存时,表现出Na+抑制Mg2+诱导的切割活性,切割效率明显低于Mg2+单独存在时的结果.这些结果提示,在生理环境下,Mg2+对于多核酶系统对底物的切割反应是必需的,而Na+则不是.     

Effect and mechanism of the Ang-(1-7) on human mesangial cells injury induced by low density lipoprotein

Hyperlipidemia is an independent risk factor for renal disease, and lipid deposition is associated with glomerulosclerosis. The angiotensin converting enzyme 2-angiotensin-(1-7)-Mas axis (ACE2-Ang-(1-7)-Mas axis) has been reported to participate in lipid metabolic regulation but its mechanism remains unclear. We hypothesized Ang-(1-7) would reduce lipid uptake in human mesangial cells (HMCs) by regulating the low density lipoprotein receptor–sterol regulatory element binding proteins 2–SREBP cleavage activating protein (LDLr–SREBP2–SCAP) negative feedback system, and improve glomerulosclerosis by regulating the transforming growth factor-β1 (TGF-β1). In this study we found that ACE2 was undetected in HMCs. The administration of LDL caused normal LDLr–SREBPs–SCAP negative feedback effect. Exogenous Ang-(1-7) enhanced this negative feedback effect via down-regulating LDLr, SREBP2, and SCAP expression, and effectively inhibited LDL-induced lipid deposition and cholesterol increases. This enhanced inhibitory effect was reversed by the Mas receptor antagonist A-779. Meanwhile, Ang-(1-7) significantly decreased the high LDL-induced production of TGF-β1, an effect blocked by A-779. Interestingly, HMCs treated with Ang-(1-7) alone activated the TGF-β1 expression. Our results suggested that Ang-(1-7) inhibits LDL accumulation and decreases cholesterol levels via modulating the LDLr–SREBPs–SCAP negative feedback system through the Mas receptor. Moreover, Ang-(1-7) exhibits a dual regulatory effect on TGF-β1 in HMCs.     

Induction of prophage lambda does not require full induction of RecA protein synthesis

In mitomycin C-treated lambda lysogens, even though the rate of synthesis of RecA protein was greatly reduced by a low concentration of rifampicin (4 microgram/ml), induction of prophage lambda occurred readily as assessed by (i) cell lysis of the lysogens, (ii) production of progeny phage, and (iii) extensive cleavage of lambda repressor. The extent and the rate of cleavage of lambda repressor were not significantly affected by the low rate of synthesis of RecA protein resulting from rifampicin action. However, the yield of phage progeny was reduced and lysis of the cells was slightly delayed. We conclude that in RecA+ bacteria, induction of prophage lambda does not require full induction of RecA protein synthesis.     

Insertion of dibasic residues directs a constitutive protein to the regulated secretory pathway.

The mechanisms for sorting proteins to the regulated secretory pathway (RSP) remains poorly understood. We recently reported that dibasic sequences that are cleaved by pro-protein convertases (PCs) in pro-neurotensin also acted as sorting signal for the precursor. Here we addressed two questions regarding the role of dibasics as sorting signal: (i) Are dibasics sufficient to direct proteins to the RSP? (ii) Do they sort proteins by virtue of their interaction with PCs? The first question was studied by inserting dibasics in beta-lactamase, a constitutively secreted protein and comparing the regulated secretion of beta-lactamase to that of its mutant in transfected endocrine cells. The second question was investigated by comparing the regulated release of pro-neurotensin in PC12 cells that are devoid of PCs to that in PC1- and PC2-transfected PC12 cells. The data show that the mutant beta-lactamase was indeed targeted in part to the RSP and that pro-neurotensin was sorted to the RSP without the assistance of the PCs, thus indicating that dibasics can act as sorting signal by themselves independently of their interaction with PCs.     

New versatile cloning and sequencing vectors based on bacteriophage M13

A new pair of cloning and sequencing vectors based on bacteriophage M13mp7 has been developed. These vectors (M13tg130 and M13tg131) contain, in addition to the EcoRI, BamHI, HindIII, SmaI, SalI and PstI sites present in other vectors [cf., M13mp8 and M13mp9, Messing and Vieira, Gene 19 (1982) 269-276], unique restriction recognition sequences for the enzymes EcoRV, KpnI, SphI, SstI and XbaI. A restriction site for the enzyme BglII has been incorporated into the polylinker region of one of the vector pair to permit rapid discrimination between the two vectors.     

HIV-1 Protease Uses Bi-Specific S2/S2′ Subsites to Optimize Cleavage of Two Classes of Target Sites

Retroviral proteases (PRs) have a unique specificity that allows cleavage of sites with or without a P1′ proline. A P1′ proline is required at the MA/CA cleavage site due to its role in a post-cleavage conformational change in the capsid protein. However, the HIV-1 PR prefers to have large hydrophobic amino acids flanking the scissile bond, suggesting that PR recognizes two different classes of substrate sequences. We analyzed the cleavage rate of over 150 combinations of six different HIV-1 cleavage sites to explore rate determinants of cleavage. We found that cleavage rates are strongly influenced by the two amino acids flanking the amino acids at the scissile bond (P2–P1/P1′–P2′), with two complementary sets of rules. When P1′ is proline, the P2 side chain interacts with a polar region in the S2 subsite of the PR, while the P2′ amino acid interacts with a hydrophobic region of the S2′ subsite. When P1′ is not proline, the orientations of the P2 and P2′ side chains with respect to the scissile bond are reversed; P2 residues interact with a hydrophobic face of the S2 subsite, while the P2′ amino acid usually engages hydrophilic amino acids in the S2′ subsite. These results reveal that the HIV-1 PR has evolved bi-functional S2 and S2′ subsites to accommodate the steric effects imposed by a P1′ proline on the orientation of P2 and P2′ substrate side chains. These results also suggest a new strategy for inhibitor design to engage the multiple specificities in these subsites.     

新型断裂内含肽介导的可控性C端断裂

内含肽介导的蛋白质断裂被广泛地应用于蛋白质纯化、连接和环化. 但目前的方法都是用传统的连续的内含肽来介导蛋白质断裂反应,因而往往存在自发性断裂、产率低等问题. 本实验选择3个S1型新型断裂内含肽Ter ThyX、Ssp GryB和Rma DnaB来实现蛋白质断裂反应的可控性. 在可控性C端断裂反应中,S1型断裂内含肽的C端片段（IC ）与硫氧还蛋白（T）融合作为前体蛋白,加入化学合成的Ssp DnaB S1型断裂内含肽 的N端小肽与二硫苏糖醇（DTT）共同诱导C端断裂反应.结果表明,该小肽可以诱导这 3个不同的S1型断裂内含肽的前体蛋白发生C端断裂反应. 该方法为利用内含肽C端断 裂介导的蛋白质纯化提供了更多的选择,并为内含肽的结构与功能的关系研究提供-有用的线索.     

Development of substrate analogue inhibitors for the human airway trypsin-like protease HAT

A series of substrate analogue inhibitors of the serine protease HAT, containing a 4-amidinobenzylamide moiety as the P1 residue, was prepared. The most potent compounds possess a basic amino acid in the d-configuration as P3 residue. Whereas inhibitor 4 (K_i 13 nM) containing proline as the P2 residue completely lacks selectivity, incorporation of norvaline leads to a potent inhibitor (15, K_i 15 nM) with improved selectivity for HAT in comparison to the coagulation proteases thrombin and factor Xa or the fibrinolytic plasmin. Selected inhibitors were able to suppress influenza virus replication in a HAT-expressing MDCK cell model.