核糖核苷酸还原酶研究

核糖核苷酸还原酶广泛存在于各种生物中,是生物体内唯一的催化4种核糖核苷酸还原、生成相应的脱氧核糖核苷酸的酶。该酶是DNA合成和修复的关键酶和限速酶,对细胞的增殖和分化起着调控作用。不同生物中的RR根据其结合的金属辅助因子不同而分类。虽然不同类型RR之间的氨基酸序列相似性很低,但它们有十分相似的三级结构的活性中心和相同的催化功能。RR分子中包含2个变构位点,即酶活性中心和底物特异结合位点。活性中心通过生物有机自由基的作用催化核糖核苷酸还原;底物特异结合位点通过变构作用调控4种dNTPs在细胞内的平衡。因此,该酶不仅是研究DNA合成与修复、细胞增殖与分化及癌症的治疗与抗癌药物开发的重要靶点,同时也是研究酶的结构与功能以及酶的催化机理等的重要工具。本文总结了该酶的种类与分布、结构特征、催化机理及作为抗癌药物开发靶点等方面的研究进展。     

Structure of a serpin-protease complex shows inhibition by deformation

The serpins have evolved to be the predominant family of serine-protease inhibitors in man. Their unique mechanism of inhibition involves a profound change in conformation, although the nature and significance of this change has been controversial. Here we report the crystallographic structure of a typical serpin-protease complex and show the mechanism of inhibition. The conformational change is initiated by reaction of the active serine of the protease with the reactive centre of the serpin. This cleaves the reactive centre, which then moves 71 A to the opposite pole of the serpin, taking the tethered protease with it. The tight linkage of the two molecules and resulting overlap of their structures does not affect the hyperstable serpin, but causes a surprising 37% loss of structure in the protease. This is induced by the plucking of the serine from its active site, together with breakage of interactions formed during zymogen activation. The disruption of the catalytic site prevents the release of the protease from the complex, and the structural disorder allows its proteolytic destruction. It is this ability of the conformational mechanism to crush as well as inhibit proteases that provides the serpins with their selective advantage.     

Crystallographic study of a beta-lactam inhibitor complex with elastase at 1.84 A resolution

The continuing discovery and development of beta-lactams as antibiotics has had an unparalleled impact on the overall health and well-being of society. Recently, appropriately substituted cephalosporins were shown to be potent inhibitors of elastase, suggesting a novel therapeutic role for the beta-lactams in the control of emphysema and other degenerative diseases. We have now solved and partially refined at atomic resolution the structure of a complex of porcine pancreatic elastase with the time-dependent irreversible inhibitor 3-acetoxymethyl-7-alpha-chloro-3-cephem-4-carboxylate-1,1-dioxide tert-butyl ester (I), the most potent of the beta-lactam elastase inhibitors yet reported. (Porcine pancreatic elastase is a close relative of the desired drug target, human polymorphonuclear leukocyte elastase.) A mechanism of action is presented, based on the structure and on biochemical evidence (T.-Y.L. et al., in preparation), which clarifies the operational similarities and differences between beta-lactam elastase inhibitors and antibiotics. Features of the reaction include the expulsion of a leaving group at the cephalosporin 3' position and the formation of two covalent bonds with the active site of porcine pancreatic elastase at residues Ser 195 and His 57.     

Solution structure of the major binding protein for the immunosuppressant FK506

The major FK506 binding protein (FKBP, relative molecular mass approximately 11,800; Mr 11.8K) and cyclophilin (Mr approximately 17K) belong to a class of proteins termed immunophilins. Although unrelated at the amino-acid sequence level, they both possess peptidyl-prolyl cis-trans isomerase activities which are inhibited by immunosuppressants that block signal transduction pathways leading to T-lymphocyte activation. FK506 and rapamycin strongly inhibit the peptidyl-prolyl cis-trans isomerase activity of FKBP, whereas cyclosporin A inhibits that of cyclophilin. The significance of this enzyme activity and the role of the immunophilins in immunoregulation is unknown. To understand better the function of the immunophilins and their interaction with inhibitors, we are investigating the solution structures of FKBP and FKBP-inhibitor complexes by multidimensional NMR methods. Here we report the solution conformation of FKBP, as generated by NMR, distance geometry and molecular dynamics methods. The regular secondary structure of FKBP is composed mainly of beta sheet (approximately 35%) with little helical structure (less than 10%). The hydrophobic core of the molecule, containing the buried side chains of six of the protein's nine aromatic amino acids, is enclosed by a five-stranded antiparallel beta sheet on one side, a loop and a short helix at residues 51-56 and 57-65, and an aperiodic loop at residues 81-95. Examination of the structure suggests a possible site of interaction with FK506.     

新型1,5-二芳基咪唑类制剂的分子对接研究

采用PM5半经验量子化学方法对29个新型1,5-二芳基咪唑类选择性环氧合酶(COX-2)抑制剂的结构进行了全优化,将这些优化结构与COX-2的活性位点进行了分子对接。对接研究表明：活性1,5－二芳基咪唑类COX-2抑制剂具有类似塞来昔布等三环类环氧合酶-2选择性抑制剂的立体结构,对接自由能与抑制剂活性有较好的相关性。     

HDACs link the DNA damage response, processing of double-strand breaks and autophagy

Protein acetylation is mediated by histone acetyltransferases (HATs) and deacetylases (HDACs), which influence chromatin dynamics, protein turnover and the DNA damage response. ATM and ATR mediate DNA damage checkpoints by sensing double-strand breaks and single-strand-DNA-RFA nucleofilaments, respectively. However, it is unclear how acetylation modulates the DNA damage response. Here we show that HDAC inhibition/ablation specifically counteracts yeast Mec1 (orthologue of human ATR) activation, double-strand-break processing and single-strand-DNA-RFA nucleofilament formation. Moreover, the recombination protein Sae2 (human CtIP) is acetylated and degraded after HDAC inhibition. Two HDACs, Hda1 and Rpd3, and one HAT, Gcn5, have key roles in these processes. We also find that HDAC inhibition triggers Sae2 degradation by promoting autophagy that affects the DNA damage sensitivity of hda1 and rpd3 mutants. Rapamycin, which stimulates autophagy by inhibiting Tor, also causes Sae2 degradation. We propose that Rpd3, Hda1 and Gcn5 control chromosome stability by coordinating the ATR checkpoint and double-strand-break processing with autophagy.     

Structural evidence for gene duplication in the evolution of the acid proteases.

X-ray studies of acid proteases indicate a bilobal structure with a well defined active site cleft. An intramolecular twofold symmetry axis relates two topologically similar domains and the active site residues. A possible mechanism for evolution by gene duplication, divergence and gene fusion is presented.     

去乙酰化酶抑制剂TSA对间充质干细胞C3H10T1/2增殖和成脂分化的影响

采用MTT和流式细胞术分别检测不同浓度的TSA对C3H10T1/2细胞活性和细胞周期分布的影响;油红O染色检测TSA对其成脂分化的影响,实时定量PCR检测TSA对成脂分化的关键转录因子PPAR-γ,以及成脂分化标志物Fabp4和Adipoq mRNA转录的影响.研究去乙酰化酶抑制剂TSA对间充质干细胞C3H10T1/2增殖和成脂分化的影响及其可能的作用机制.结果显示TSA浓度为1、10和30 nmol/L呈浓度依赖性地抑制C3H10T1/2细胞活性,改变细胞形态,并将其细胞周期抑制在G0/G1期;TSA浓度为10nmol/L明显抑制C3H10T1/2细胞的成脂分化作用,并呈浓度依赖性地抑制PPAR-γ、Fabp4和Adipoq mRNA的转录.表明TSA呈剂量依赖性地抑制间充质干细胞C3H10T1/2的增殖和成脂分化,除转录水平调控外,非组蛋白如细胞骨架相关蛋白可能也参与TSA的抑制作用.     

青霉菌组蛋白去乙酰化酶基因的敲除及其次级代谢产物变化

丝状真菌尤其是青霉菌Penicillium代谢的各类次级产物日趋成为研制新药的重要来源,很多药物如抗癌药物、抗生素、免疫抑制剂等均来源于真菌。然而真菌次级代谢受多因素的影响,其中表观遗传修饰起到重要的调控作用。组蛋白乙酰化表观遗传修饰常与转录激活相关,从而促进次级代谢产物的合成。以青霉属真菌Penicillium christenseniae SD-193.84为研究对象,利用生物信息学手段确定其组蛋白去乙酰化酶（HDAC）基因,建立该菌中同源重组基因敲除技术,对该基因进行敲除,并比较了基因敲除前后次级代谢产物的变化,发现HDAC影响了多种次级代谢产物的合成。本研究为青霉菌分子遗传操作及次级代谢调控提供了参考。     

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.     

腈基类化合物对半胱氨酸蛋白酶的抑制机理

半胱氨酸蛋白酶是人体自身产生的重要酶类物质,它广泛的参与人体新陈代谢和蛋白的水解。但过量产生的半胱氨酸蛋白酶会导致骨质疏松和乳腺癌。腈基类化合物分子是一种新发现的具有抑制半胱氨酸蛋白酶作用的靶向药物分子,高效且低副作用。但是它的抑制机理一直没有得到解决。本文介绍借助量子力学/分子力学（Q M/M M）的计算研究,解得了此类化合物对于半胱氨酸蛋白酶的抑制机理,并借助分子学软件设计了一种新型药物分子,有助进一步药物研究。     

Structure of the catalytic domain of the hepatitis C virus NS2-3 protease

Hepatitis C virus is a major global health problem affecting an estimated 170 million people worldwide. Chronic infection is common and can lead to cirrhosis and liver cancer. There is no vaccine available and current therapies have met with limited success. The viral RNA genome encodes a polyprotein that includes two proteases essential for virus replication. The NS2-3 protease mediates a single cleavage at the NS2/NS3 junction, whereas the NS3-4A protease cleaves at four downstream sites in the polyprotein. NS3-4A is characterized as a serine protease with a chymotrypsin-like fold, but the enzymatic mechanism of the NS2-3 protease remains unresolved. Here we report the crystal structure of the catalytic domain of the NS2-3 protease at 2.3 A resolution. The structure reveals a dimeric cysteine protease with two composite active sites. For each active site, the catalytic histidine and glutamate residues are contributed by one monomer, and the nucleophilic cysteine by the other. The carboxy-terminal residues remain coordinated in the two active sites, predicting an inactive post-cleavage form. Proteolysis through formation of a composite active site occurs in the context of the viral polyprotein expressed in mammalian cells. These features offer unexpected insights into polyprotein processing by hepatitis C virus and new opportunities for antiviral drug design.