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1.
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. 相似文献
2.
Interactions of serine proteases with cultured fibroblasts 总被引:1,自引:0,他引:1
D D Cunningham W E Van Nostrand D H Farrell C H Campbell 《Journal of cellular biochemistry》1986,32(4):281-291
This review summarizes the mechanisms by which several serine proteases, particularly urokinase, thrombin, and elastase, interact with cultured fibroblasts. Many of these studies were prompted by findings that interactions of these proteases with cells and the extracellular matrix are important in a number of physiologic and pathologic processes. Two main pathways have been identified for specific interactions of these proteases with fibroblasts. One involves surface binding sites for the free protease that appear to bind only one particular protease. An unusual feature collectively shared by the binding sites for urokinase, thrombin, and elastase is that the bound protease is not detectably internalized by the fibroblasts. The other pathway by which serine proteases interact with fibroblasts involves proteins named protease nexins (PNs). Three PNs have been identified. They are secreted by fibroblasts and inhibit certain serine proteases by forming a covalent complex with the protease catalytic site serine. The complexes then bind back to the fibroblasts via the PN portion of the complex and are internalized and degraded. Recent studies showing that the fibroblast surface and extracellular matrix accelerate the inactivation of thrombin by PN-1 support the hypothesis that the PNs control protease activity at and near the cell surface. The PNs differ from plasma protease inhibitors in their molecular properties, absence in plasma, site of synthesis, and site of clearance of the inhibitor:protease complexes. 相似文献
3.
Understanding the regulation of physiological processes requires detailed knowledge of the recognition of substrates by enzymes. One of the most productive model systems for the study of enzyme-substrate interactions is the serine protease family; however, most studies of protease action have used small substrates that contain an activated, non-natural scissile bond. Because few kinetic or structural studies have used protein substrates, the physiologically relevant target of most proteases, it seems likely that important mechanisms of substrate recognition and processing by proteases have not yet been fully elucidated. Consistent with this hypothesis, we have observed that K(m) values for protein substrates are reduced as much as 200-15000-fold relative to those of analogous peptide substrates. Here we examine the thermodynamic consequences of interactions between proteases and their substrates using staphylococcal nuclease (SNase) and SNase variants as model protein substrates. We have obtained values for enthalpy, entropy, and K(d) for binding of proteins and peptides by the nonspecific protease trypsin and the highly specific protease urokinase-type plasminogen activator (u-PA). To avoid cleavage of substrates during these measurements, we used inactive variants of trypsin and u-PA whose catalytic serine S195 had been replaced by alanine. Differences in the K(d) values for binding of protein and peptide substrates closely approximate the large differences observed in the corresponding K(m) values. Improved binding of protein substrates is due to decreased enthalpy, and this effect is pronounced for the selective protease u-PA. Fundamental differences in recognition of analogous protein and peptide substrates may have influenced the evolution of protease specificity. 相似文献
4.
We have used ultrasonic velocimetry, high-precision densimetry, and fluorescence spectroscopy, in conjunction with isothermal titration and differential scanning calorimetry, to characterize the binding of turkey ovomucoid third domain (OMTKY3) to alpha-chymotrypsin. We report the changes in volume and adiabatic compressibility that accompany the association of these proteins at 25 degrees C and pH 4.5. In addition, we report the changes in free energy, enthalpy, entropy, and heat capacity upon the binding of OMTKY3 to alpha-chymotrypsin over a temperature range of 20-40 degrees C. Our volume and compressibility data, in conjunction with X-ray crytsallographic data on the OMTKY3-alpha-chymotrypsin complex, suggest that 454(+/-22) water molecules are released to the bulk state upon the binding of OMTKY3 to alpha-chymotrypsin. Furthermore, these volumetric data suggest that the intrinsic compressibility of the two proteins decreases by 7%. At each temperature studied, OMTKY3 association with alpha-chymotrypsin is entropy driven with a large, unfavorable enthalpy contribution. The observed entropy of the binding reflects interplay between two very large favorable and unfavorable terms. The favorable term reflects an increase in the hydrational entropy resulting from release to the bulk of 454 water molecules. The unfavorable term is related to a decrease in the configurational entropy and, consequently, a decrease in the conformational dynamics of the two proteins. In general, we discuss the relationship between macroscopic and microscopic properties, in particular, identifying and quantifying the role of hydration in determining the thermodynamics of protein recognition as reflected in volumetric and calorimetric parameters. 相似文献
5.
Edgcomb SP Baker BM Murphy KP 《Protein science : a publication of the Protein Society》2000,9(5):927-933
The heat of binding the serine protease, porcine pancreatic elastase, by the inhibitor, turkey ovomucoid third domain, is dependent on the presence of inorganic phosphate. This dependence is saturable and can be accurately modeled as the phosphate binding to a single site on the protease-inhibitor complex; thus, the elastase-ovomucoid system provides a unique opportunity to study phosphate-protein interactions. We have used isothermal titration calorimetry to investigate this binding, thereby providing one of the few complete thermodynamic characterizations of phosphate interacting with proteins. The binding is characterized by a small favorable deltaG degrees, a large unfavorable deltaH degrees, and a positive deltaCp, thermodynamics consistent with the release of water being linked to phosphate binding. These measurements provide insight into the binding of phosphotyrosine containing peptides to SH2 domains by suggesting the energetic consequences of binding phosphate free from other interactions. 相似文献
6.
The blotched snakehead virus (BSNV), an aquatic birnavirus, encodes a polyprotein (NH2-pVP2-X-VP4-VP3-COOH) that is processed through the proteolytic activity of its own protease (VP4) to liberate itself and the viral proteins pVP2, X and VP3. The protein pVP2 is further processed by VP4 to give rise to the capsid protein VP2 and four structural peptides. We report here the crystal structure of a VP4 protease from BSNV, which displays a catalytic serine/lysine dyad in its active site. This is the first crystal structure of a birnavirus protease and the first crystal structure of a viral protease that utilizes a lysine general base in its catalytic mechanism. The topology of the VP4 substrate binding site is consistent with the enzymes substrate specificity and a nucleophilic attack from the si-face of the substrates scissile bond. Despite low levels of sequence identity, VP4 shows similarities in its active site to other characterized Ser/Lys proteases such as signal peptidase, LexA protease and Lon protease. Together, the structure of VP4 provides insights into the mechanism of a recently characterized clan of serine proteases that utilize a lysine general base and reveals the structure of potential targets for antiviral therapy, especially for other related and economically important viruses, such as infectious bursal disease virus in poultry and infectious pancreatic necrosis virus in aquaculture. 相似文献
7.
【背景】丝氨酸蛋白酶在木霉菌生物防治过程中发挥重要作用。【目的】研究绿木霉丝氨酸蛋白酶S8/S53超家族基因信息及其生物学功能,进而为该蛋白酶生防制剂的开发及基因改造提供理论支持。【方法】通过生物信息学分析方法,从绿木霉Gv29-8基因组中鉴定出23个丝氨酸蛋白酶基因,以少孢节丛孢菌ATCC 24927基因组中鉴定的4个丝氨酸蛋白酶基因作为对照,对这27个丝氨酸蛋白酶基因的特性、蛋白结构、进化地位、功能等进行预测分析。【结果】27个基因结构差异较大,编码的蛋白具有典型的丝氨酸蛋白酶催化三联体结构,属于S8/S53超家族,分为6个亚家族,同一亚家族的蛋白酶保守区长度相近,相似性较高,催化残基附近序列比较保守。系统进化分析显示,同一亚家族丝氨酸蛋白酶聚为一类。【结论】绿木霉和少孢节丛孢菌的部分丝氨酸蛋白酶基因在结构和蛋白性质上相似性强,亲缘关系较近,均属于S8PCSK9ProteinaseKlike亚家族,推测绿木霉与少孢节丛孢菌该亚家族的丝氨酸蛋白酶具有相似的功能,可抑制植物病原真菌和降解线虫体壁。 相似文献
8.
Bateman KS Huang K Anderson S Lu W Qasim MA Laskowski M James MN 《Journal of molecular biology》2001,305(4):839-849
X-ray crystallography has been used to determine the 3D structures of two complexes between Streptomyces griseus proteinase B (SGPB), a bacterial serine proteinase, and backbone variants of turkey ovomucoid third domain (OMTKY3). The natural P1 residue (Leu18I) has been substituted by a proline residue (OMTKY3-Pro18I) and in the second variant, the peptide bond between Thr17I and Leu18I was replaced by an ester bond (OMTKY3-psi[COO]-Leu18I). Both variants lack the P1 NH group that donates a bifurcated hydrogen bond to the carbonyl O of Ser214 and O(gamma) of the catalytic Ser195, one of the common interactions between serine proteinases and their canonical inhibitors. The SGPB:OMTKY3-Pro18I complex has many structural differences in the vicinity of the S1 pocket when compared with the previously determined structure of SGPB:OMTKY3-Leu18I. The result is a huge difference in the DeltaG degrees of binding (8.3 kcal/mol), only part of which can be attributed to the missing hydrogen bond. In contrast, very little structural difference exists between the complexes of SGPB:OMTKY3-psi[COO]-Leu18I and SGPB:OMTKY3-Leu18I, aside from an ester O replacing the P1 NH group. Therefore, the difference in DeltaG degrees, 1.5 kcal/mol as calculated from the measured equilibrium association constants, can be attributed to the contribution of the P1 NH hydrogen bond toward binding. A crystal structure of OMTKY3 having a reduced peptide bond between P1 Leu18I and P'1 Asp19I, (OMTKY3-psi[CH2NH2+]-Asp19I) has also been determined by X-ray crystallography. This variant has very weak association equilibrium constants with SGPB and with chymotrypsin. The structure of the free inhibitor suggests that the reduced peptide bond has not introduced any major structural changes in the inhibitor. Therefore, its poor ability to inhibit serine proteinases is likely due to the disruptions of the canonical interactions at the oxyanion hole. 相似文献
9.
丝氨酸蛋白酶抑制剂Ea的表达纯化与活性分析 总被引:1,自引:0,他引:1
Ea是一种植物来源的丝氨酸蛋白酶抑制剂,分子量为18kD。利用其与丝氨酸蛋白酶家族成员的结合特性,可用于丝氨酸蛋白酶的结构与功能研究,也可作为亲和层析的配体而用于丝氨酸蛋白酶的纯化。将Ea基因插入大肠杆菌表达载体pET11a,在BL21(DE3)菌中以包涵体形式表达出重组蛋白质,表达量可占菌体蛋白质总量的30%。将包涵体变性、复性,得到具有天然抑制活性的rEa。经两步纯化所得rEa的纯度达到967%以上。活性分析表明,rEa对胰蛋白酶和人组织型纤溶酶原激活剂均有抑制作用。制备成rEaSepharose亲和柱可有效结合胰蛋白酶。 相似文献
10.
Sequence-to-reactivity algorithms (SRAs) for proteins have the potential of being broadly applied in molecular design. Recently, Laskowski et al. have reported an additivity-based SRA that accurately predicts most of the standard free energy changes of association for variants of turkey ovomucoid third domain (OMTKY3) with six serine peptidases, one of which is streptogrisin B (commonly known as Streptomyces griseus peptidase B, SGPB). Non-additivity effects for residues 18I and 32I, and for residues 20I and 32I of OMTKY3 occurred when the associations with SGPB were predicted using the SRA. To elucidate precisely the mechanics of these non-additivity effects in structural terms, we have determined the crystal structures of the unbound OMTKY3 (with Gly32I as in the wild-type amino acid sequence) at a resolution of 1.16 A, the unbound Ala32I variant of OMTKY3 at a resolution of 1.23 A, and the SGPB:OMTKY3-Ala32I complex (equilibrium association constant K(a)=7.1x10(9) M(-1) at 21(+/-2) C degrees, pH 8.3) at a resolution of 1.70 A. Extensive comparisons with the crystal structure of the unbound OMTKY3 confirm our understanding of some previously addressed non-additivity effects. Unexpectedly, SGPB and OMTKY3-Ala32I form a 1:2 complex in the crystal. Comparison with the SGPB:OMTKY3 complex shows a conformational change in the SGPB:OMTKY3-Ala32I complex, resulting from a hinged rigid-body rotation of the inhibitor caused by the steric hindrance between the methyl group of Ala32IA of the inhibitor and Pro192BE of the peptidase. This perturbs the interactions among residues 18I, 20I, 32I and 36I of the inhibitor, probably resulting in the above non-additivity effects. This conformational change also introduces residue 10I as an additional hyper-variable contact residue to the SRA. 相似文献
11.
Lu X Lamontagne J Lu F Block TM 《Apoptosis : an international journal on programmed cell death》2008,13(4):483-494
Serine protease dependent cell apoptosis (SPDCA) is a recently described caspase independent innate apoptotic pathway. It
differs from the traditional caspase dependent apoptotic pathway in that serine proteases, not caspases, are critical to the
apoptotic process. The mechanism of SPDCA is still unclear and further investigation is needed to determine any role it may
play in maintaining cellular homeostasis and development of disease. The current knowledge about this pathway is limited only
to the inhibitory effects of some serine protease inhibitors. Synthetic agents such as pefabloc, AEBSF and TPCK can inhibit
this apoptotic process in cultured cells. There is little known, however, about biologically active agents available in the
cell which can inhibit SPDCA. Here, we show that over-expression of a cellular protein called serine protease inhibitor Kazal
(SPIK/TATI/PSTI) results in a significant decrease in cell susceptibility to SPDCA, suggesting that SPIK is an apoptosis inhibitor
suppressing this pathway of apoptosis. Previous work has associated SPIK and cancer development, indicating that this finding
will help to open the doorway for further study on the mechanism of SPDCA and the role it may play in cancer development. 相似文献
12.
Common protein architecture and binding sites in proteases utilizing a Ser/Lys dyad mechanism 总被引:1,自引:0,他引:1
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Escherichia coli signal peptidase (SPase) and E. coli UmuD protease are members of an evolutionary clan of serine proteases that apparently utilize a serine-lysine catalytic dyad mechanism. Recently, the crystallographic structure of a SPase inhibitor complex was solved elucidating the catalytic residues and the substrate binding subsites. Here we show a detailed comparison of the E. coli SPase structure to the native E. coli UmuD' structure. The comparison reveals that despite a very low sequence identity these functionally diverse enzymes share the same protein fold within their catalytic core and allows by analogy for the assignment of the cleavage-site orientation and substrate binding subsites in the UmuD(D') protease. The structural alignment of SPase and UmuD' predicts important mechanistic and structural similarities and differences within these newly characterized families of serine proteases. 相似文献
13.
Tang Yongqing Nicole Drentin Renee C. Duncan Lakshmi C. Wijeyewickrema Robert N. Pike 《Biochimica et Biophysica Acta - Proteins and Proteomics》2012,1824(1):253-262
The lectin pathway of the complement system is activated following the binding of carbohydrate-based ligands by recognition molecules such as mannose-binding lectin (MBL) or ficolins. Engagement of the recognition molecules causes activation of associated MBL-associated serine proteases or MASPs, which in turn activate downstream complement molecules to activate the system. Two MASP genes are alternatively spliced during expression to yield 5 proteins, including three proteases (MASP-1, -2 and -3) and two truncated proteins, MAp19 and MAp44. Here we discuss what is currently known about these proteins in terms of their structure and function. MASP-2 is autoactivated following the initial binding events of the pathway and is able to subsequently activate the C4 and C2 substrates required to activate the rest of the pathway. MASP-1 is able to augment MASP-2 activation, but also appears to play other roles, although the physiological significance of these is not yet clear. The roles of the truncated Map19 and Map44 proteins and the MASP-3 protease are currently unknown. The proteases form an interesting sub-family of proteins that clearly should be the focus of future research in order to establish their biological roles.This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome. 相似文献
14.
M. E. Popov M. A. Sten’gach N. S. Andreeva 《Russian Journal of Bioorganic Chemistry》2008,34(3):380-386
A three-dimensional structure of histo-aspartic protease (HAP), a pepsin-like enzyme from the causative agent of malaria Plasmodium falciparum, is suggested on the basis of homologous modeling followed by equilibration by the method of molecular dynamics. The presence of a His residue in the catalytic site instead of an Asp residue, which is characteristic of pepsin-like enzymes, and replacement of some other conserved residues in the active site make it possible for the enzyme to function by the covalent mechanism inherent in serine proteases. The detailed structures of HAP complexes with pepstatin, a noncovalent inhibitor of aspartic proteases, and phenylmethylsulfonyl fluoride, a covalent inhibitor of serine proteases, as well as with a pentapeptide substrate are discussed. 相似文献
15.
Carvalho AL Sanz L Barettino D Romero A Calvete JJ Romão MJ 《Journal of molecular biology》2002,322(2):325-337
Prostate-specific kallikrein, a member of the gene family of serine proteases, was initially discovered in semen and is the most useful serum marker for prostate cancer diagnosis and prognosis. We report the crystal structure at 1.42A resolution of horse prostate kallikrein (HPK). This is the first structure of a serine protease purified from seminal plasma. HPK shares extensive sequence homology with human prostate-specific antigen (PSA), including a predicted chymotrypsin-like specificity, as suggested by the presence of a serine residue at position S1 of the specificity pocket. In contrast to other kallikreins, HPK shows a structurally distinct specificity pocket. Its entrance is blocked by the kallikrein loop, suggesting a possible protective or substrate-selective role for this loop. The HPK structure seems to be in an inactivated state and further processing might be required to allow the binding of substrate molecules. Crystal soaking experiments revealed a binding site for Zn(2+) and Hg(2+), two known PSA inhibitors. 相似文献
16.
Morris MT Cheng WC Zhou XW Brydges SD Carruthers VB 《International journal for parasitology》2004,34(6):693-701
Serine protease inhibitors have been implicated in viral and parasite pathogenesis through their ability to inhibit apoptosis, provide protection against digestive enzymes in the gut and dictate host range specificity. Two Kazal family serine protease inhibitors from the obligate intracellular parasite Toxoplasma gondii (TgPI-1 and TgPI-2) have been characterised previously. Here, we describe the identification and initial characterisation of a novel Kazal inhibitor, NcPI-S, from a closely related apicomplexan parasite, Neospora caninum. Unlike the multidomain inhibitors identified in T. gondii, NcPI-S is a single domain inhibitor bearing a methionine in the position (P1) that typically dictates specificity for target proteases. Based on this, NcPI-S was predicted to inhibit elastase, chymotrypsin and subtilisin. However, we found that recombinant NcPI-S inhibited subtilisin very well, with little or no activity against elastase or chymotrypsin. NcPI-S localises to the dense granules and is secreted into the parasitophorous vacuole. Finally, antibodies raised against recombinant NcPI-S recognise two polypeptides in an N. caninum lysate, one with a molecular mass approximately 11 kDa and another at approximately 20 kDa. This, along with mass spectrometry analysis of recombinant NcPI-S, suggests that the inhibitor is expressed as a dimer in the parasite. 相似文献
17.
Mujun Yu Goutam Chakraborty Michael Grabow Nicholas A. Ingoglia 《Neurochemical research》1994,19(1):105-110
The tRNA mediated, posttranslational, N-terminal arginylation of proteins occurs in all eukaryotic cells. In nervous tissue, these reactions can be inhibited by endogenous molecules with a molecular weight of between one thousand and five thousand. In the present experiments, exogenous serine protease inhibitors (10–5M or less) but not other types of protease inhibitors, were found to be able to block the arginylation of protein in extracts of rat brain homogenates. Inhibition was not by the usual mode of action of protease inhibitors, but by interfering (non-competitively) with the charging of tRNA. Since arginylated proteins are rapidly ubiquitinated and degraded by cytosolic proteases, serine protease inhibitors may act to stabilize proteins by a dual mechanism of inhibiting arginylation as well as inhibiting serine proteases. 相似文献
18.
Runyon ST Zhang Y Appleton BA Sazinsky SL Wu P Pan B Wiesmann C Skelton NJ Sidhu SS 《Protein science : a publication of the Protein Society》2007,16(11):2454-2471
High-temperature requirement A (HtrA) and its homologs contain a serine protease domain followed by one or two PDZ domains. Bacterial HtrA proteins and the mitochondrial protein HtrA2/Omi maintain cell function by acting as both molecular chaperones and proteases to manage misfolded proteins. The biological roles of the mammalian family members HtrA1 and HtrA3 are less clear. We report a detailed structural and functional analysis of the PDZ domains of human HtrA1 and HtrA3 using peptide libraries and affinity assays to define specificity, structural studies to view the molecular details of ligand recognition, and alanine scanning mutagenesis to investigate the energetic contributions of individual residues to ligand binding. In common with HtrA2/Omi, we show that the PDZ domains of HtrA1 and HtrA3 recognize hydrophobic polypeptides, and while C-terminal sequences are preferred, internal sequences are also recognized. However, the details of the interactions differ, as different domains rely on interactions with different residues within the ligand to achieve high affinity binding. The results suggest that mammalian HtrA PDZ domains interact with a broad range of hydrophobic binding partners. This promiscuous specificity resembles that of bacterial HtrA family members and suggests a similar function for recognizing misfolded polypeptides with exposed hydrophobic sequences. Our results support a common activation mechanism for the HtrA family, whereby hydrophobic peptides bind to the PDZ domain and induce conformational changes that activate the protease. Such a mechanism is well suited to proteases evolved for the recognition and degradation of misfolded proteins. 相似文献
19.
Proteolytic cleavage of a limited number of cellular proteins is a central biochemical feature of apoptosis. Aspartate-specific cysteine proteases, the so-called caspases, are the main enzymes involved in this process. At least ten homologues of interleukin-1 converting enzyme (ICE), the first described human caspase, have been identified so far. The purified active proteins are heterodimers with a long and a short subunit derived from a common inactive precursor. Crystallized ICE has an original tetrameric structure. The various caspases tend to show high degrees of homology around the active site Cys. Proteolysis by caspases minimally requires a tetrapeptide substrate in which Asp is an absolute requirement in P1 position, the P4 substrate residue is unique to each homologue, and much more widespread amino acid substitution is observed in P2 and P3. Caspase activation might involve a proteolytic cascade similar to that of the coagulation cascade but the molecular ordering of these proteases in vivo remains to be established clearly. Calpains, serine proteases, granzymes and the proteasome–ubiquitin pathway of protein degradation are other proteolytic pathways that have been suggested to play a role in apoptosis. Substrate proteins can be either activated or degraded during cell death and the consequences of their cleavage remains mostly ill-understood. Nevertheless, the recent demonstration that protease inhibitors can rescue mice undergoing acute liver destruction indicates the accuracy of therapeutic strategies aiming to inhibit cell death-associated proteolysis. 相似文献
20.
Variants of the serine protease, subtilisin BPN', in which the catalytic triad residues (Ser-221, His-64, and Asp-32) are replaced singly or in combination by alanine retain activities with the substrate N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (sAAPF-pna) that are at least 10(3) to 10(4) above the non-enzymatic rate [Carter, P., Wells, J.A. Nature (London) 322:564-568, 1988]. A possible source of the residual activity was the hydrogen bond with the N delta 2 of Asn-155 that helps to stabilize the oxyanion generated in the tetrahedral transition state during amide bond hydrolysis by the wild-type enzyme. Replacing Asn-155 by Gly (N155G) lowers the turnover number (kcat) for sAAPF-pna by 150-fold with virtually no change in the Michaelis constant (KM). However, upon combining the N155G and S221A mutations to give N155G:S221A, kcat is actually 5-fold greater than for the S221A enzyme. Thus, the catalytic role of Asn-155 is dependent upon the presence of Ser-221. The residual activity of the N155G:S221A enzyme (approximately 10(4)-fold above the uncatalyzed rate) is not an artifact because it can be completely inhibited by the third domain of the turkey ovomucoid inhibitor (OMTKY3), which forms a strong 1:1 complex with the active site. The mutations N155G and S221A individually weaken the interaction between subtilisin and OMTKY3 by 1.8 and 2.0 kcal/mol, respectively, and in combination by 2.1 kcal/mol. This is consistent with disruption of stabilizing interactions around the reactive site carbonyl of the OMTKY3 inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献