丝氨酸蛋白酶抑制剂B9与相关免疫细胞

丝氨酸蛋白酶抑制剂B9(Serpin B9)是蛋白酶抑制剂超家族成员,包括人丝氨酸蛋白酶抑制剂(PI-9)及鼠丝氨酸蛋白酶抑制剂同源蛋白(SPI-6),是主要针对颗粒蛋白酶B(GrB)的内源性蛋白酶抑制剂.Serpin B9的调节在T淋巴细胞、树突状细胞、自然杀伤细胞、中性粒细胞、间充质干细胞、肿瘤细胞有抑制凋亡、维持细胞平衡、调节免疫反应、增强DNA疫苗能力等功能.研究Serpin B9的作用机理,将有利于相关免疫疾病发生机制的探索,并为临床干细胞治疗、抗肿瘤治疗等提供新的理论依据.     

α_1-抗胰蛋白酶和丝氨酸蛋白酶抑制剂——变异和反变异

人的血浆含有几种蛋白酶抑制剂,这些蛋白酶抑制剂约占血浆总蛋白含量的10%.如广谱抑制剂α_2-巨球蛋白即为重要一例.但这类抑制剂中研究最多、血浆含量最高的是具有特异性的α_1-抗胰蛋白酶,虽然它可以抑制大部分的丝氨酸蛋白酶,但它主要是抑制弹性蛋白酶特别是抑制嗜中性白细胞释放的弹性蛋白酶.现已证明α_1-抗胰蛋白酶是一族新的同源蛋白的主要成员.这一族同源蛋白可以抑制丝氨酸蛋白酶,故统称为丝氨酸蛋白酶抑制剂(serpin).研究α_1-抗胰蛋白酶所获     

组织因子途径抑制物-2的结构及其生物学作用研究进展

蛋白水解失调是机体许多病理过程的主要特征，例如癌症、动脉粥样硬化和炎症等。蛋白酶抑制剂在血液凝集、补体固定、纤维蛋白溶解、受精和胚胎形成等多种生物学过程中均发挥重要的调控作用。大部分的蛋白酶抑制剂都具有多肽支架的特征，包括Kunitz家族、Kazal家族、Serpin家族和Mucus家族等，其中Kunitz家族即丝氨酸蛋白酶抑制剂家族由20多个成员组成，主要包括牛胰蛋白酶抑制剂（bovine pancreatic trypsin inhibitor，BPTI）、组织因子途径抑制物（tissue factor pathway inhibitor，TFPI-1）及其同系物TFPI-2等，     

重组纤溶酶抑制剂Textilinin-1蛋白含量测定方法的对比研究

正Textilinin-1是从拟眼镜蛇Pseudonajatextilistextilis中提取的Kunitz型蛋白酶抑制剂,具有纤溶酶抑制活性,可抑制基于纤溶酶被激活导致纤维蛋白溶解所导致的出血,因此具有开发为止血药的潜力,可以作为外科手术中抑肽酶的替代药物~([1])。目前已有含Textilinin-1基因的重组毕赤酵母菌种,能高效表达目的蛋白~([2])。蛋白质含量测定是重组蛋白类药物质量控制中的重要     

人类组织因子途径抑制因子的研究进展

人类组织因子途径抑制因子属于库尼(Kunitz)型丝氨酸蛋白酶抑制剂家族蛋白,分为组织因子途径抑制因子-1(tissuefactorpathwayinhibitor-1,TFPI-1)和组织因子途径抑制因子-2(tissuefactorpathwayinhibitor-2,TFPI-2)。TFPI-1以抗凝血作用为主,而TFPI-2是广谱丝氨酸蛋白酶抑制剂。二者在结构上有部分同源性,均由3个重复的Kunitz结构域组成。但二者在基因序列、组织来源、分布和作用机理上的很大差异,导致二者在多种生理和病理过程中发挥的作用也大不相同。就有关研究进展做一综述。     

弹性蛋白酶及其在肺损伤中的作用

弹性蛋白酶的作用底物极广,弹性蛋白酶及其抑制剂的失平衡使弹性蛋白酶有损伤正常组织的可能性。影响蛋白酶抑制剂的各种因子对弹性蛋白酶损伤作用有重要影响。本文分下述几部分进行介绍。 一、正常人体弹性蛋白酶的分类及特点 (一)根据酶的作用特点分为丝氨酸蛋白酶和金属蛋白酶两大类,丝氨酸蛋白酶类主要有中性粒细胞弹性蛋白酶、胰弹性蛋白酶等。金属蛋白酶类弹性蛋白酶主要有巨噬细胞弹性蛋白酶等。 (二)根据弹性蛋白酶产生的部位,可分以下几种:1.胰弹性蛋白酶:为丝氨酸蛋白     

丙型肝炎病毒丝氨酸蛋白酶及抑制剂研究进展

丙型肝炎病毒(heptitis cvirus,HCV)丝氨酸蛋白酶(serine protease)是催化病毒前体蛋白中非结构蛋白部分裂解的关键蛋白酶.对病毒复制和宿主细胞都有重要作用,是当前抗病毒研究的一个重要靶点.本文从丝氨酸蛋白酶结构、功能和抑制剂等三方面,介绍丝氨酸蛋白酶的研究进展,这对该酶抑制研究和丙型肝炎的治疗研究会有积极意义.     

丙型肝炎病毒丝氨酸蛋白酶及抑制剂研究进展

丙型肝炎病毒(heptitis cvirus，HCV)丝氨酸蛋白酶(serine protease)是催化病毒前体蛋白中非结构蛋白部分裂解的关键蛋白酶。对病毒复制和宿主细胞都有重要作用，是当前抗病毒研究的一个重要靶点。本文从丝氨酸蛋白酶结构、功能和抑制剂等三方面，介绍丝氨酸蛋白酶的研究进展，这对该酶抑制研究和丙型肝炎的治疗研究会有积极意义。     

α_1-抗胰蛋白酶和丝氨酸蛋白酶抑制剂——变异和反变异

人的血浆有几种蛋白酶抑制剂,这些蛋白酶抑制剂约占血浆总蛋白含量的10％。如广谱抑制剂α_2-巨球蛋白即为重要一例。但这类抑制剂中研究最多、血浆含量最高的是具有特异性的α_1-抗胰蛋白酶,虽然它可以抑制大部分的丝氨酸蛋白酶,但它主要是抑制弹性蛋白酶特别是抑制嗜中性白细胞释放的弹性蛋白酶。α_1-抗胰蛋白酶的功能是通过其基因缺陷导致结缔组织过早破坏使肺失去弹性引起肺气肿的过程而了解的。这种基因缺陷分子病理的广泛研究最近在完成正常和变异分子结构的研究时达到了高峰。现已证明α_1-抗胰蛋白酶是一族新的同源蛋白     

肿瘤相关胰蛋白酶抑制剂的研究概况

肿瘤相关胰蛋白酶抑制剂(tumor-associated trypsin inhibitor,TATI)又被称为胰腺分泌胰蛋白酶抑制剂(pancreatic secretory trypsin inhibitor,PSTI)或丝氨酸蛋白酶抑制因子Kazal type 1型(SPINK1),是一种由56个氨基酸残基组成的分泌性多肽,主要作用是抑制胰蛋白酶等多种丝氨酸蛋白酶活性.除此之外,还具有一些其他作用,如类生长因子作用、拮抗细胞杀伤作用等.TAT I在肿瘤中可以作为一种生物标志物.本文将对现阶段关于TAT I在肿瘤中作用的相关热点研究进行综述.     

Human U937 cell surface peptidase activities: characterization and degradative effect on tumor necrosis factor-alpha.

Surface peptidase activities on the human monocytic lineage cell line U937 were characterized. Two diisopropyl phosphofluoridate (DFP)-inhibitable serine peptidases were identified by differences in their hydrolytic activities on chromogenic peptides: one removed tripeptides from the free NH2-terminal end of the synthetic peptide Ala-Ala-Phe-p-nitroanilide (pNA) and was not inhibited by inhibitors of metallo-, cysteic-, and aspartic-proteinases, or by those of elastase-, trypsin- and chymotrypsin-like enzymes, suggesting the presence of a hitherto unidentified serine tripeptidyl endopeptidase; the other peptidase catalyzed the release of Gly-Pro from Gly-Pro-pNA and was inhibited by DFP, phenylmethyl sulfonyl fluoride and diprotin A, thus resembling dipeptidyl peptidase IV (DPP IV) with respect to its substrate specificity and inhibitor profile. A group of N-exo-aminopeptidase activities specifically inhibited by bestatin, was also detected when Ala-, Leu-, Arg- and Lys-pNA were used a substrates. The activities were surface associated and not secreted as determined by extracellular location of product and enzymatic recovery in highly purified U937 cell membranes. Peripheral monocytes and macrophages were found to virtually exhibit identical levels of these two classes of peptidase activities when compared to those detected on U937 cells. The relative contributions of these hydrolytic enzymes to the cleavage of bioactive and radioiodinated cytokines including tumor necrosis factor-alpha (TNF-alpha), interleukin-1 alpha and interferon-gamma was next examined. The results indicated that N-aminopeptidases do not appear to participate in the catabolism of any tested cytokine. In contrast, the most interesting finding was that both serine peptidases participate in TNF-alpha degradation. Analysis of the final proteolytic digestion products demonstrated the disappearance of the native 17-kDa molecule TNF-alpha, and the concomitant release of biologically inactive fragments of less than or equal to 2 kDa. Together, these observations indicate new roles for both the DPP IV-like enzyme and the tripeptidyl endopeptidase located at the surface of human monocytic cells, including the regulation of the extracellular TNF-alpha concentration. Thus, the identification of functional ectopeptidases provides insight into their potential role in both normal and malignant monocytic function.     

Pseudallescheria boydii releases metallopeptidases capable of cleaving several proteinaceous compounds

Pseudallescheria boydii is an opportunistic filamentous fungus that causes serious infections in humans. Virulence attributes expressed by P. boydii are unknown. Conversely, peptidases are incriminated as virulence factors in several pathogenic fungi. Here we investigated the extracellular peptidase profile in P. boydii. After growth on Sabouraud for 7 days, mycelia of P. boydii were incubated for 20 h in PBS-glucose. The cell-free PBS-glucose supernatant was submitted to SDS-PAGE and 12 secretory polypeptides were observed. Two of these polypeptides (28 and 35 kD) presented proteolytic activity when BSA was used as a copolymerized substrate. The extracellular peptidases were most active in acidic pH (5.5) and fully inhibited by 1,10-phenanthroline, a zinc-metallopeptidase inhibitor. Other metallo-, cysteine, serine and aspartic proteolytic inhibitors did not significantly alter these activities. To confirm that these enzymes belong to the metallo-type peptidases, the apoenzymes were obtained by dialysis against chelating agents, and supplementation with different cations, especially Cu(2+) and Zn(2+), restored their activities. Except for gelatin, both metallopeptidases hydrolyzed various co-polymerized substrates, including human serum albumin, casein, hemoglobin and IgG. Additionally, the metallopeptidases were able to cleave different soluble proteinaceous substrates such as extracellular matrix components and sialylated proteins. All these hydrolyses were inhibited by 1,10-phenanthroline. Interestingly, Scedosporium apiospermum (the anamorph of P. boydii) produced a distinct extracellular peptidase profile. Collectively, our results demonstrated for the first time the expression of acidic extracellular metallopeptidases in P. boydii capable of degrading several proteinaceous compounds that could help the fungus to escape from natural human barriers and defenses.     

Secretory aspartyl peptidase activity from mycelia of the human fungal pathogen Fonsecaea pedrosoi: effect of HIV aspartyl proteolytic inhibitors

Fonsecaea pedrosoi is the principal causative agent of chromoblastomycosis, which is a chronic, often debilitating, suppurative and granulomatous mycosis. Very little is known about the hydrolytic enzymes produced by this human fungal pathogen. In the present study, we have identified extracellular proteolytic activity from F. pedrosoi mycelial forms when grown in chemically defined conditions. Secretory aspartyl peptidase activity was measured during 15 days of fungal growth in vitro using bovine serum albumin (BSA) as the soluble substrate and extreme acidic pH (2.0). This activity was totally inhibited by pepstatin A, a classic aspartyl peptidase inhibitor. Conversely, metallo (o-phenanthroline), cysteine (E-64) and serine (PMSF) proteolytic inhibitors failed to restrain proteolytic activity. We also evaluated the effect of four distinct HIV aspartyl peptidase inhibitors on the secretory proteolytic activity of F. pedrosoi mycelia. Indinavir, ritonavir and nelfinavir powerfully inhibited extracellular aspartyl proteolytic activity by approximately 97, 96 and 87%, respectively, whereas saquinavir did not significantly interfere with BSA hydrolysis. Mycelial-derived secretory aspartyl peptidase activity cleaved other proteinaceous substrates, including human albumin, fibrinogen, fibronectin, laminin and type I collagen. As previously reported by our group, conidia also produce secretory aspartyl peptidase. In this sense, we investigated the effect of pepstatin A on F. pedrosoi development. Pepstatin A was able to inhibit the growth of conidium and its transformation into mycelium. Taken together, our results suggest a possible participation of aspartyl peptidases in the essential fungal processes, such as growth, differentiation, nutrition and cleavage of relevant host proteinaceous components.     

Fibroblast activation protein-α promotes tumor growth and invasion of breast cancer cells through non-enzymatic functions

Fibroblast activation protein-α (FAP) is a cell surface, serine protease of the post-prolyl peptidase family that is expressed in human breast cancer but not in normal tissues. Previously, we showed that FAP expression increased tumor growth rates in a mouse model of human breast cancer. Here the role of the proteolytic activities of FAP in promoting tumor growth, matrix degradation and invasion was investigated. Mammary fat pads of female SCID mice were inoculated with breast cancer cells that express FAP and the mice treated with normal saline or Val-boroPro (talabostat); Glu-boroPro (PT-630); or 1-[[(3-hydroxy-1-adamantyl)amino]acetyl]-2-cyano-(S)-pyrrolidine (LAF-237) that inhibit prolyl peptidases. Other mice were injected with breast cancer cells expressing a catalytically inactive mutant of FAP and did not receive inhibitor treatment. PT-630 and LAF-237 did not slow growth of tumors produced by any of the three cell lines expressing FAP. Talabostat slightly decreased the growth rates of the FAP-expressing tumors but because PT-630 and LAF-237 did not, the growth retardation was likely not related to the inhibition of FAP or the related post-prolyl peptidase dipeptidyl peptidase IV. Breast cancer cells expressing a catalytically inactive mutant of FAP (FAP^S624A) also produced tumors that grew rapidly. In vitro studies revealed that cells expressing wild type FAP or FAP^S624A degrade extracellular matrix (ECM) more extensively, accumulate higher levels of matrix metalloproteinase-9 (MMP-9) in conditioned medium, are more invasive in type I collagen gels, and have altered signaling compared to control transfectants that do not express FAP and form slow growing tumors. We conclude that the proteolytic activity of FAP participates in matrix degradation, but other functions of the protein stimulate increased tumor growth.     

Cathepsin G degrades denatured collagen

Neutrophils are known to contain several metalloproteinases that can damage collagen, a major structural component of the extracellular matrix. Here a neutrophil serine proteinase secreted from activated neutrophils was shown to cleave denatured collagen (gelatin). This serine proteinase was not inhibited by synthetic inhibitors of elastase (elastatinal or Me-O-suc-Ala-Pro-Val-CH₂Cl). However, a synthetic inhibitor of cathepsin G (Z-Gly-Leu-Phe-CH₂Cl) was able to inhibit the serine proteinase having gelatinolytic activity, indicating that cathepsin G, a major serine proteinase, from neutrophils is responsible for cleaving gelatin. Purified cathepsin G was also shown to degrade gelatin. In further experiments, oxidized glutathione was able to enhance the gelatinolytic activity of cathepsin G. These results show that cathepsin G is capable of cleaving denatured collagen, and its activity is enhanced or stabilized in the presence of gtutathione. The data support the concept that cathepsin G released from neutrophils could play a major role in degrading collagen during inflammation and may in part account for the degradation of extracellular matrix during inflammation.     

Cyclic dimeric GMP signaling regulates intracellular aggregation, sessility, and growth of Ehrlichia chaffeensis

Cyclic dimeric GMP (c-di-GMP), a bacterial second messenger, is known to regulate bacterial biofilm and sessility. Replication of an obligatory intracellular pathogen, Ehrlichia chaffeensis, is characterized by formation of bacterial aggregates called morulae inside membrane-bound inclusions. When E. chaffeensis matures into an infectious form, morulae become loose to allow bacteria to exit from host cells to infect adjacent cells. E. chaffeensis expresses a sensor kinase, PleC, and a cognate response regulator, PleD, which can produce c-di-GMP. A hydrophobic c-di-GMP antagonist, 2'-O-di(tert-butyldimethysilyl)-c-di-GMP (CDGA) inhibits E. chaffeensis internalization into host cells by facilitating degradation of some bacterial surface proteins via endogenous serine proteases. In the present study, we found that PleC and PleD were upregulated synchronously during exponential growth of bacteria, concomitant with increased morula size. While CDGA did not affect host cells, when infected cells were treated with CDGA, bacterial proliferation was inhibited, morulae became less compact, and the intracellular movement of bacteria was enhanced. Concurrently, CDGA treatment facilitated the extracellular release of bacteria with lower infectivity than those spontaneously released from sham-treated cells. Addition of CDGA to isolated inclusions induced dispersion of the morulae, degradation of an inclusion matrix protein TRP120, and bacterial intrainclusion movement, all of which were blocked by a serine protease inhibitor. These results suggest that c-di-GMP signaling regulates aggregation and sessility of E. chaffeensis within the inclusion through stabilization of matrix proteins by preventing the serine protease activity, which is associated with bacterial intracellular proliferation and maturation.     

Alpha1-antitrypsin inhibits the activity of the matriptase catalytic domain in vitro

Matriptase is a type II transmembrane protease that is characterized by an N-terminal transmembrane and multiple extracellular domains, in addition to the conserved extracellular serine protease catalytic domain. The expression pattern of matriptase suggests that this protease may play broad roles in the biology of surface lining epithelial cells. In this study we report that alpha1-antitrypsin (AAT), an endogenous inhibitor of serine proteases, inhibits the catalytic domain of human recombinant matriptase in vitro. Co-incubation of AAT with matriptase (at a molar ratio 1:2) resulted in the formation of heat stable complexes, clearly seen in sodium dodecyl sulfate electrophoresis and Western blots. AAT was found to be a slow, tight-binding inhibitor of the catalytic domain of matriptase with a second order reaction rate constant of 0.31 x 10(3) M(-1)s(-1). Notably, the oxidized form of AAT, which lacks serine protease inhibitor activity, failed to generate matriptase complexes and to inhibit matriptase activity. Since matriptase is involved in a number of physiologic processes, including activation of epithelial sodium channels, our findings offer considerable new insights into new regulatory function of AAT in vivo.     

Role of a serine endopeptidase in the hydrolysis of exogenous cholecystokinin by brain slices

The participation of a serine endopeptidase, previously shown to be involved in endogenous cholecystokinin inactivation [Rose, Camus and Schwartz (1989) Neuroscience 29, 583-594], in the hydrolysis of various exogenous cholecystokinin peptides was studied with slices from rat cerebral cortex. In order to protect intermediate fragments from further degradation and mimick experimental conditions in this previous study, most experiments were performed in the presence of Thiorphan, an enkephalinase inhibitor, and bestatin, an aminopeptidase inhibitor, which did not significantly affect the rate of cholecystokinin-8 hydrolysis. All peptide fragments formed after incubation of cholecystokinin-8, non-sulphated cholecystokinin-8, cholecystokinin-6, cholecystokinin-5, cholecystokinin-4 or Asp-Tyr-Met-Gly-Trp were identified by isocratic high-performance liquid chromatography in several systems, fluorescence spectra and/or amino acid analysis. When identified, the appearing fragments were quantified by u.v. spectrophotometry and found to fully account for the substrate disappearance. The hydrolysis rate was higher for short cholecystokinin peptides than for the octapeptide and was, in all cases, diminished by 30-50% in the presence of diisopropyl fluorophosphate, a serine peptidase inhibitor. One of the main hydrolysis products of cholecystokinin-8, or its non-sulphated analogue, was cholecystokinin-5, whose formation was impaired in the presence of diisopropyl fluorophosphate. Cholecystokinin-5 itself was apparently a substrate for a serine peptidase leading to the formation of the tripeptide Gly-Trp-Met, later cleaved into Trp-Met and Trp. Hence a serine endopeptidase(s) appears to be responsible for cleavage of the two peptides bonds of the cholecystokinin-8 molecule where the carboxyl group is donated by a methionine residue.2+n addition,