叉角厉蝽丝氨酸蛋白酶及抑制剂基因的克隆与表达分析

为明确叉角厉蝽Eocanthecona furcellata (Wolff)丝氨酸蛋白酶基因EfSP1及抑制剂基因EfSPI20的基因序列特征和时空转录特征,为其生理功能研究奠定基础。利用PCR克隆技术获得叉角厉蝽唾液腺EfSPI20和EfSP1的完整开放阅读框(Open reading frame, ORF)序列,使用生物信息学软件进行序列分析以及系统进化分析,采用实时荧光定量PCR (Real time quantitativate PCR,RT-qPCR)分析两个基因分别在叉角厉蝽不同发育时期和组织中的表达特征。结果表明,EfSPI20与EfSP1基因完整开放阅读框长度分别为378 bp和921 bp,分别编码125个氨基酸和306个氨基酸,预测均为亲水蛋白质,理论分子量分别为13.48 kDa和33.82 kDa,等电点分别为6.68和5.80,分别有30个和23个氨基酸残基的信号肽序列,EfSPI20有跨膜结构域,EfSP1无跨膜结构域。序列比对显示叉角厉蝽EfSPI20与茶翅蝽Halyomorpha halys PPI同源性最高,氨基酸序列一致性达58%;EfSP1与稻绿蝽Nezara viridula SP同源性最高,氨基酸序列一致性达66%;系统发育树显示叉角厉蝽与同为蝽科的茶翅蝽和稻绿蝽物种亲缘关系近。EfSPI20基因在雌雄成虫和唾液腺中高表达,推测EfSPI20可能具有抑制胰蛋白酶活性的功能和与叉角厉蝽的捕食消化相关;EfSP1基因在卵期、卵巢和肠道中高表达,推测EfSP1可能与叉角厉蝽的生殖功能和蛋白消化相关。     

Specificity and reactive loop length requirements for crmA inhibition of serine proteases

The viral serpin, crmA, is distinguished by its small size and ability to inhibit both serine and cysteine proteases utilizing a reactive loop shorter than most other serpins. Here, we characterize the mechanism of crmA inhibition of serine proteases and probe the reactive loop length requirements for inhibition with two crmA reactive loop variants. P1 Arg crmA inhibited the trypsin-like proteases, thrombin, and factor Xa, with moderate efficiencies (approximately 10(2)-10(4) M(-1)sec(-1)), near equimolar inhibition stoichiometries, and formation of SDS-stable complexes which were resistant to dissociation (k(diss) approximately 10(-7) sec(-1)), consistent with a serpin-type inhibition mechanism. Trypsin was not inhibited, but efficiently cleaved the variant crmA as a substrate (k(cat)/K(M) of approximately 10(6) M(-1) sec(-1)). N-terminal sequencing confirmed that the P1 Arg-P1'Cys bond was the site of cleavage. Altering the placement of the Arg in a double mutant P1 Gly-P1'Arg crmA resulted in minimal ability to inhibit any of the trypsin family proteases. This variant was cleaved by the proteases approximately 10-fold less efficiently than P1 Arg crmA. Surprisingly, pancreatic elastase was rapidly inhibited by wild-type and P1 Arg crmAs (10(5)-10(6) M(-1)sec(-1)), although with elevated inhibition stoichiometries and higher rates of complex dissociation. N-terminal sequencing showed that elastase attacked the P1'Cys-P2'Ala bond, indicating that crmA can inhibit proteases using a reactive loop length similar to that used by other serpins, but with variations in this inhibition arising from different effective P2 residues. These results indicate that crmA inhibits serine proteases by the established serpin conformational trapping mechanism, but is unusual in inhibiting through either of two adjacent reactive sites.     

Thrombin inhibitor design: X-ray and solution studies provide a novel P1 determinant

The crystal structures of proflavin and 6-fluorotryptamine thrombin have been completed showing binding of both ligands at the active site S1 pocket. The structure of proflavin:thrombin was confirmatory, while the structure of 6-fluorotryptamine indicated a novel binding mode at the thrombin active site. Furthermore, speculation that the sodium atom identified in an extended solvent channel beneath the S1 pocket may play a role in binding of these ligands was investigated by direct proflavin titrations as well as chromogenic activity measurements as a function of sodium concentration at constant ionic strength. These results suggested a linkage between the sodium site and the S1 pocket. This observation could be due to a simple ionic interaction between Asp189 and the sodium ion or a more complicated structural rearrangement of the thrombin S1 pocket. Finally, the unique binding mode of 6-fluorotryptamine provides ideas toward the design of a neutrally charged thrombin inhibitor.     

Recombinant hirustasin: production in yeast,crystallization, and interaction with serine proteases.

A synthetic gene coding for the 55-amino acid protein hirustasin, a novel tissue kallikrein inhibitor from the leech Hirudo medicinalis, was generated by polymerase chain reaction using overlapping oligonucleotides, fused to the yeast alpha-factor leader sequence and expressed in Saccharomyces cerevisiae. Recombinant hirustasin was secreted mainly as incompletely processed fusion protein, but could be processed in vitro using a soluble variant of the yeast yscF protease. The processed hirustasin was purified to better than 97% purity. N-terminal sequence analysis and electrospray ionization mass spectrometry confirmed a correctly processed N-terminus and the expected amino acid sequence and molecular mass. The biological activity of recombinant hirustasin was identical to that of the authentic leech protein. Crystallized hirustasin alone and in complex with tissue kallikrein diffracted beyond 1.4 A and 2.4 A, respectively. In order to define the reactive site of the inhibitor, the interaction of hirustasin with kallikrein, chymotrypsin, and trypsin was investigated by monitoring complex formation in solution as well as proteolytic cleavage of the inhibitor. During incubation with high, nearly equimolar concentration of tissue kallikrein, hirustasin was cleaved mainly at the peptide bond between Arg 30 and Ile 31, the putative reactive site, to yield a modified inhibitor. In the corresponding complex with chymotrypsin, mainly uncleaved hirustasin was found and cleaved hirustasin species accumulated only slowly. Incubation with trypsin led to several proteolytic cleavages in hirustasin with the primary scissile peptide bond located between Arg 30 and Ile 31. Hirustasin appears to fall into the class of protease inhibitors displaying temporary inhibition.     

Dimers initiate and propagate serine protease inhibitor polymerisation

The serine protease inhibitor (serpin) family can readily form long-chain polymers by a process that underlies a variety of diseases. We show here that monomers of plasma serpins α₁-antitrypsin and antithrombin are stable on incubation with the rate-limiting step in their polymerisation being the formation of the initial dimer. Once formed, the dimers readily interlink to form tetramers and can bind monomers to form trimers and longer oligomers. Cleavage of the only exposed reactive loop, in unit I of the dimers, prevents their interlinkage, but these cleaved dimers can still link to monomers. The rapid binding by the cleaved dimers of a peptide specific to the lower half of β-sheet A of the molecule indicates the ready opening of this β-sheet in unit II of the dimers. The failure of the cleaved dimers to bind peptide-complexed monomers, together with the relative inaccessibility of the P14 hinge residue in the oligomers, is evidence that partial insertion of the reactive loop into its own A-sheet is required for polymer formation. We propose that serpin dimers initiate and propagate polymerisation by having one exposed loop with an optimal conformation as a β-strand donor and a readily opened β-sheet as an acceptor. The sequential reformation of these activated β-interfaces as the oligomer extends, molecule by molecule, provides a model for the fibril and amyloid formation of conformational diseases in general as well as for the infectivity of prion encephalopathies.     

Structure–activity relationship of marinostatin,a serine protease inhibitor isolated from a marine organism

A 12‐residue MST isolated from a marine organism is a potent serine protease inhibitor that has a double cyclic structure composed of two ester linkages formed between the β‐hydroxyl and β‐carboxyl groups, Thr³‐Asp⁹ and Ser⁸‐Asp¹¹. MST was synthesized by a regioselective esterification procedure employing two sets of orthogonally removable side‐chain protecting groups for the Asp and Ser/Thr residues. In the MST molecule, there were no significant changes observed in yield by changing the order of esterification. SAR study of MST revealed that the minimum required structure for expressing the inhibitory activity is the sequence (1–9) in a monocyclic structure where Pro⁷ located in the ring plays a crucial role in keeping the structural rigidity. By applying the structural motif of MST, we rationally designed protease inhibitory specificities that differ from those of the natural product. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Crystal structures of uninhibited factor VIIa link its cofactor and substrate-assisted activation to specific interactions

Factor VIIa initiates the extrinsic coagulation cascade; this event requires a delicately balanced regulation that is implemented on different levels, including a sophisticated multi-step activation mechanism of factor VII. Its central role in hemostasis and thrombosis makes factor VIIa a key target of pharmaceutical research. We succeeded, for the first time, in recombinantly producing N-terminally truncated factor VII (rf7) in an Escherichia coli expression system by employing an oxidative, in vitro, folding protocol, which depends critically on the presence of ethylene glycol. Activated recombinant factor VIIa (rf7a) was crystallised in the presence of the reversible S1-site inhibitor benzamidine. Comparison of this 1.69A crystal structure with that of an inhibitor-free and sulphate-free, but isomorphous crystal form identified structural details of factor VIIa stimulation. The stabilisation of Asp189-Ser190 by benzamidine and the capping of the intermediate helix by a sulphate ion appear to be sufficient to mimic the disorder-order transition conferred by the cofactor tissue factor (TF) and the substrate factor X. Factor VIIa shares with the homologous factor IXa, but not factor Xa, a bell-shaped activity modulation dependent on ethylene glycol. The ethylene glycol-binding site of rf7a was identified in the vicinity of the 60 loop. Ethylene glycol binding induces a significant conformational rearrangement of the 60 loop. This region serves as a recognition site of the physiologic substrate, factor X, which is common to both factor VIIa and factor IXa. These results provide a mechanistic framework of substrate-assisted catalysis of both factor VIIa and factor IXa.     

Protease inhibitors derived from elafin and SLPI and engineered to have enhanced specificity towards neutrophil serine proteases

The secretory leukocyte protease inhibitor (SLPI), elafin, and its biologically active precursor trappin‐2 are endogeneous low‐molecular weight inhibitors of the chelonianin family that control the enzymatic activity of neutrophil serine proteases (NSPs) like elastase, proteinase 3, and cathepsin G. These inhibitors may be of therapeutic value, since unregulated NSP activities are linked to inflammatory lung diseases. However SLPI inhibits elastase and cathepsin G but not proteinase 3, while elafin targets elastase and proteinase 3 but not cathepsin G. We have used two strategies to design polyvalent inhibitors of NSPs that target all three NSPs and may be used in the aerosol‐based treatment of inflammatory lung diseases. First, we fused the elafin domain with the second inhibitory domain of SLPI to produce recombinant chimeras that had the inhibitory properties of both parent molecules. Second, we generated the trappin‐2 variant, trappin‐2 A62L, in which the P1 residue Ala is replaced by Leu, as in the corresponding position in SLPI domain 2. The chimera inhibitors and trappin‐2 A62L are tight‐binding inhibitors of all three NSPs with subnanomolar K_is, similar to those of the parent molecules for their respective target proteases. We have also shown that these molecules inhibit the neutrophil membrane‐bound forms of all three NSPs. The trappin‐2 A62L and elafin‐SLPI chimeras, like wild‐type elafin and trappin‐2, can be covalently cross‐linked to fibronectin or elastin by a tissue transglutaminase, while retaining their polypotent inhibition of NSPs. Therefore, the inhibitors described herein have the appropriate properties to be further evaluated as therapeutic anti‐inflammatory agents.     

重组人丝氨酸蛋白酶抑制因子Hespintor Kazal结构域的原核表达、纯化及活性鉴定

Hespintor是应用抑制消减杂交技术 (SSH) 从肝母细胞瘤细胞系HepG2中筛选得到的一未知功能蛋白,序列分析表明该蛋白属于Kazal型丝氨酸蛋白酶抑制因子 (Serine proteinase inhibitor, Serpin) 家族中的一个分泌型新成员,具有与食管癌相关基因2 (Esophageal cancer related gene 2, ECRG2) 高度同源的Serpin基本结构。为了进一步阐明Hespintor的生物学功能,必须得到纯化的Hespintor蛋白。先将Hespintor Kazal结构域编码序列亚克隆至原核表达载体pET-40b(+),转化至Rosetta (DE3) 表达宿主菌中。经 0.25 mmol/L IPTG,30 ℃诱导5 h获得了分子量约为42 kDa的Hespintor-Kazal重组融合蛋白的优化表达,Western blotting证实了重组蛋白的特异性。Hespintor-Kazal重组融合蛋白以包涵体形式在宿主菌中表达,利用金属螯合亲和层析和阴离子交换层析柱对重组蛋白进行两步纯化。初步的活性鉴定表明,纯化的Hespintor-Kazal重组融合蛋白能特异性抑制胰蛋白酶的水解活性,提示Hespintor具有作为一种新型抗肿瘤药物的潜在开发价值。     

Interaction of Kazal-type inhibitor domains with serine proteinases: biochemical and structural studies

The interaction of domains of the Kazal-type inhibitor protein dipetalin with the serine proteinases thrombin and trypsin is studied. The functional studies of the recombinantly expressed domains (Dip-I+II, Dip-I and Dip-II) allow the dissection of the thrombin inhibitory properties and the identification of Dip-I as a key contributor to thrombin/dipetalin complex stability and its inhibitory potency. Furthermore, Dip-I, but not Dip-II, forms a complex with trypsin resulting in an inhibition of the trypsin activity directed towards protein substrates. The high resolution NMR structure of the Dip-I domain is determined using multi-dimensional heteronuclear NMR spectroscopy. Dip-I exhibits the canonical Kazal-type fold with a central alpha-helix and a short two-stranded antiparallel beta-sheet. Molecular regions essential for inhibitor complex formation with thrombin and trypsin are identified. A comparison with molecular complexes of other Kazal-type thrombin and trypsin inhibitors by molecular modeling shows that the N-terminal segment of Dip-I fulfills the structural prerequisites for inhibitory interactions with either proteinase and explains the capacity of this single Kazal-type domain to interact with different proteinases.     

Protamine: a unique and potent inhibitor of oligopeptidase B.

Oligopeptidase B is a serine endopeptidase found in prokaryotes, unicellular eukaryotes and higher plants. The enzyme has been shown recently to play a central role in the pathogenesis of several parasitic diseases such as African trypanosomiasis, and to be a potential therapeutic target. This study reports that protamine, a basic peptide rich in arginine, is a potent inhibitor at the nanomolar level of oligopeptidase B from E. coli and wheat. Protamines 1B, 2C, 3A and TP17 displayed similar inhibitory activities and were capable of binding strongly to oligopeptidase B without proteolytic cleavage. The concentration of protamine needed for 50% inhibition (IC50) of oligopeptidase B was 10(4)-fold lower than the IC50 of trypsin. Oligopeptidase B was highly sensitive to inhibition by protamines even in the presence of serum (IC50, 1 microM). These data indicate that protamines might provide information useful for the design of more specific synthetic oligopeptidase B inhibitors.     

Isolation and characterization of a protease inhibitor from spinach leaves

An acid-stable and heat-labile proteinous protease inhibitor which was found in spinach leaves but not in seeds was isolated by sequential chromatography and preparative isoelectric focusing. The isoelectric point of this inhibitor was 4.5. The inhibitor had a M_r of ca 18 000 and was rich in aspartic acid and glycine; it had 4 half-cystine, 2 tryptophan and no methionine residues. Its extinction coefficient (E|_cm^%) was 13.7 at 280 nm. The inhibition was competitive and the dissociation constant was 3.32 × 10^?13 M. The inhibitor was specific to serine proteases and strongly inhibited trypsin and weakly inhibited α-chymotrypsin and kallikrein.     

Reconciling the lock-and-key and dynamic views of canonical serine protease inhibitor action

The efficiency of canonical serine protease inhibitors is conventionally attributed to the rigidity of their protease binding loop with no conformational change upon enzyme binding, yielding an example of the lock-and-key model for biomolecular interactions. However, solution-state structural studies revealed considerable flexibility in their protease binding loop. We resolve this apparent contradiction by showing that enzyme binding of small, 35-residue inhibitors is actually a dynamic conformer selection process on the nanosecond-timescale. Thus, fast timescale dynamics enables the association rate to be solely diffusion-controlled just like in the rigid-body model.     

Comparison of anionic and cationic trypsinogens: the anionic activation domain is more flexible in solution and differs in its mode of BPTI binding in the crystal structure

Unlike bovine cationic trypsin, rat anionic trypsin retains activity at high pH. This alkaline stability has been attributed to stabilization of the salt bridge between the N-terminal Ile16 and Asp194 by the surface negative charge (Soman K, Yang A-S, Honig B, Fletterick R., 1989, Biochemistry 28:9918-9926). The formation of this salt bridge controls the conformation of the activation domain in trypsin. In this work we probe the structure of rat trypsinogen to determine the effects of the surface negative charge on the activation domain in the absence of the Ile16-Asp194 salt bridge. We determined the crystal structures of the rat trypsin-BPTI complex and the rat trypsinogen-BPTI complex at 1.8 and 2.2 A, respectively. The BPTI complex of rat trypsinogen resembles that of rat trypsin. Surprisingly, the side chain of Ile16 is found in a similar position in both the rat trypsin and trypsinogen complexes, although it is not the N-terminal residue and cannot form the salt bridge in trypsinogen. The resulting position of the activation peptide alters the conformation of the adjacent autolysis loop (residues 142-153). While bovine trypsinogen and trypsin have similar CD spectra, the CD spectrum of rat trypsinogen has only 60% of the intensity of rat trypsin. This lower intensity most likely results from increased flexibility around two conserved tryptophans, which are adjacent to the activation domain. The NMR spectrum of rat trypsinogen contains high field methyl signals as observed in bovine trypsinogen. It is concluded that the activation domain of rat trypsinogen is more flexible than that of bovine trypsinogen, but does not extend further into the protein core.     

Insight to the residue in P2 position prevents the peptide inhibitor from being hydrolyzed by serine proteases

ABSTRACT

Peptidic inhibitors of proteases are attracting increasing interest not only as drug candidates but also for studying the function and regulation mechanisms of these enzymes. Previously, we screened out a cyclic peptide inhibitor of human uPA and found that Ala substitution of P2 residue turns upain-1 to a substrate. To further investigate the effect of P2 residue on the peptide behavior transformation, we constructed upain-1-W3F, which has Phe replacement in the P2 position. We determined K_D and K_i of upain-1-W3F and found that upain-1-W3F might still exist as an inhibitor. Furthermore, the high-resolution crystal structure of upain-1-W3F·uPA reveals that upain-1-W3F indeed stays as an intact inhibitor bind to uPA. We thus propose that the P2 residue plays a nonnegligible role in the conversion of upain-1 to a substrate. These results also proposed a strategy to optimize the pharmacological properties of peptide-based drug candidates by hydrophobicity and steric hindrance.

Abbreviations : uPA: urokinase-type plasminogen activator; SPD: serine protease domain; S1 pocket: specific substrate-binding pocket     

Anagliptin,a potent dipeptidyl peptidase IV inhibitor: its single-crystal structure and enzyme interactions

Abstract

The single-crystal structure of anagliptin, N-[2-({2-[(2S)-2-cyanopyrrolidin-1-yl]-2-oxoethyl}amino)-2-methylpropyl]-2-methylpyrazolo[1,5-a]pyrimidine-6-carboxamide, was determined. Two independent molecules were held together by intermolecular hydrogen bonds, and the absolute configuration of the 2-cyanopyrrolidine ring delivered from l-prolinamide was confirmed to be S. The interactions of anagliptin with DPP-4 were clarified by the co-crystal structure solved at 2.85?Å resolution. Based on the structure determined by X-ray crystallography, the potency and selectivity of anagliptin were discussed, and an SAR study using anagliptin derivatives was performed.     

Structure and energetics of protein-protein interactions: the role of conformational heterogeneity in OMTKY3 binding to serine proteases

Proteins with flexible binding surfaces can interact with numerous binding partners. However, this promiscuity is more difficult to understand in "rigid-body" proteins, whose binding results in little, or no, change in the position of backbone atoms. The binding of Kazal inhibitors to serine proteases is considered a classic case of rigid-body binding, although they bind to a wide range of proteases. We have studied the thermodynamics of binding of the Kazal serine protease inhibitor, turkey ovomucoid third domain (OMTKY3), to the serine protease subtilisin Carlsberg using isothermal titration calorimetry and have determined the crystal structure of the complex at very high resolution (1.1A). Comparison of the binding energetics and structure to other OMTKY3 interactions demonstrates that small changes in the position of side-chains can make significant contributions to the binding thermodynamics, including the enthalpy of binding. These effects emphasize that small, "rigid-body" proteins are still dynamic structures, and these dynamics make contributions to both the enthalpy and entropy of binding interactions.     

Structural basis for specific inhibition of the highly sensitive ShHTL7 receptor

Striga hermonthica is a root parasitic plant that infests cereals, decimating yields, particularly in sub‐Saharan Africa. For germination, Striga seeds require host‐released strigolactones that are perceived by the family of HYPOSENSITIVE to LIGHT (ShHTL) receptors. Inhibiting seed germination would thus be a promising approach for combating Striga. However, there are currently no strigolactone antagonists that specifically block ShHTLs and do not bind to DWARF14, the homologous strigolactone receptor of the host. Here, we show that the octyl phenol ethoxylate Triton X‐100 inhibits S. hermonthica seed germination without affecting host plants. High‐resolution X‐ray structures reveal that Triton X‐100 specifically plugs the catalytic pocket of ShHTL7. ShHTL7‐specific inhibition by Triton X‐100 demonstrates the dominant role of this particular ShHTL receptor for Striga germination. Our structural analysis provides a rationale for the broad specificity and high sensitivity of ShHTL7, and reveals that strigolactones trigger structural changes in ShHTL7 that are required for downstream signaling. Our findings identify Triton and the related 2‐[4‐(2,4,4‐trimethylpentan‐2‐yl)phenoxy]acetic acid as promising lead compounds for the rational design of efficient Striga‐specific herbicides.     

大豆蚜Kazal型丝氨酸蛋白酶抑制剂基因AgKaSPI对蜡蚧刺束梗孢菌侵染的表达响应

[目的]探究Kazal型丝氨酸蛋白酶抑制剂KaSPI在大豆蚜Aphis glycines的生长发育、消化和免疫防御等过程中的作用.[方法]基于大豆蚜转录组数据PCR克隆大豆蚜Kazal型丝氨酸蛋白酶抑制剂基因cDNA序列;qRT-PCR分别检测AgKaSPI在大豆蚜1-4龄若虫和成虫以及蜡蚧刺束梗孢菌Akanthomy...