Myocardial matrix metalloproteinase(s): localization and activation

Matrix metalloproteinases (MMPs) and neutrophil elastate (NE) may each contribute to fibrillar collagen degradation in various disease states. Little, however, is known about the activation and localization of MMP in the heart. Accordingly, we extracted MMP and examined mechanisms of proMMP activation in whole tissue extracts of the adult rat myocardium. Incubation of extracts with serine proteases (i.e., trypsin or neutrophil elastase) at 37°C resulted in a time-dependent activation of proMMPs. Based on immunoblot and measurements of MMP activity by zymography, the molecular weight of active MMP was deduced to be 52 kDa. The second-order rate constant for activation of proMMP by serine protease was 5.5±0.2×10⁵ M^–1min^–1 and for oxidized glutathione (GSSG) 1.5±0.1 M^–1min^–1. Incubation of the extract with both serine protease and GSSG increased the rate of activation 30-fold. Based on reverse zymographic analysis of collagenase inhibition, tissue inhibitors of metalloproteinases were identified. Indirect immunofluorescence localized proMMPs/MMPs to the endothelium and subendothelial space of the endocardium and throughout the interstitial space found between groups of muscle fibers. These results suggest that the mechanism of activation of MMPs by either a serine protease and by oxidizing, thiol-modifying reagents are mechanistically different and the presence of either a serine protease or GSSG synergistically increase the rate of activation of proMMPs. Our results also suggest that MMPs may be regulated by its own endogenous inhibitors. The contribution of this proteolytic enzyme to tissue remodeling and wound healing responses that occur in various diseases states remains to be established.Abbreviations GSSG Oxidized Glutathione - MMP Matrix Metalloproteinase - NE Neutrophil Elastase - TIMP Tissue Inhibitor of Metalloproteinase     

Isolation and Characterization of the ALP1 Protease from <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> and Its Protein Inhibitor from <Emphasis Type="Italic">Physarium polycephalum</Emphasis>

It is known that Aspergillus fumigatus secretes a serine protease ALP1 of the subtilisin family in the presence of extracellular protein substrates. We found conditions of A. fumigatus culturing that provide a high ALP1 activity inside cells without induction by extracellular proteins. The identity of the properties of the secreted and intracellular enzymes was shown. A thermostable protein inhibitor of the ALP1 protease was isolated from the plasmodium of myxomycete Physarum polycephalum. Its molecular mass is 32–33 kDa. It inhibits the ALP1 protease activity with IC₅₀ of 0.14 μM and was also shown to be a less efficient inhibitor of the activity of HIV-1 protease (IC₅₀ 2.5 μM).__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 3, 2005, pp. 259–268.Original Russian Text Copyright © 2005 by Davies, Kalinina, Samokhvalova, Malakhova, Scott, Venning, Volynskaya, Nesmeyanov.     

Kunitz型丝氨酸蛋白酶抑制剂IsKuI-1的原核表达、纯化及活性研究

目的:原核表达并制备重组蜱kunitz型丝氨酸蛋白酶抑制剂IsKuI-1,检测其抗凝血及抑制蛋白酶活性。方法:构建pET32a-sumo/IsKuI-1原核表达质粒,并转入到E. coli BL21(DE3)中,用IPTG诱导表达。表达产物经Ni-NTA亲和层析,在层析柱上用SUMO蛋白酶切割融合伴侣,纯化后得到重组目的多肽rIsKuI-1。用PT及aPTT法检测重组目的多肽的抗凝活性,发色底物法检测rIsKuI-1对丝氨酸蛋白酶的抑制活性。结果:用原核表达系统获得了rIsKuI-1,其无延长PT及aPTT活性,对人中性粒细胞弹性蛋白酶具有较好的抑制活性(IC50=1.83μM),且特异性强。结论:IsKuI-1是一种活性较好的人NE抑制剂。因此为进一步探讨rIsKuI-1的生物学功能及其作为新药开发应用奠定了基础。     

Induction of apoptosis by a calpain stimulator, ONO-3403

The effects of a synthetic serine protease inhibitor, FOY-305, and its derivatives, ONO-3403 and FO-349, on the proliferation of mouse NIH3T3 cells were investigated. At concentrations between 10 and 100 g/ml, three protease inhibitors induced a moderate suppression of cell growth. However, only ONO-3403 showed severe cytotoxicity at concentrations higher than 200 g/ml. Results of TUNEL staining and DNA fragmentation analysis indicated that ONO-3403 induced apoptosis at the high concentrations. Biochemical analysis has shown that ONO-3403 directly enhanced the amidolytic activity of purified -calpain at a concentration higher than 100 g/ml while FOY-305 and FO-349 showed less effects. When the cell extract was incubated in the presence of ONO-3403, specific degradation of a few proteins including protein kinase C was observed. Similar degradation was also observed by addition of -calpain to the extract. These results imply that ONO-3403 is a specific stimulator of calpain. It seems reasonable to conclude that increase in calpain activity results in apoptosis.This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.     

Mesotrypsin Signature Mutation in a Chymotrypsin C (CTRC) Variant Associated with Chronic Pancreatitis

Human chymotrypsin C (CTRC) protects against pancreatitis by degrading trypsinogen and thereby curtailing harmful intra-pancreatic trypsinogen activation. Loss-of-function mutations in CTRC increase the risk for chronic pancreatitis. Here we describe functional analysis of eight previously uncharacterized natural CTRC variants tested for potential defects in secretion, proteolytic stability, and catalytic activity. We found that all variants were secreted from transfected cells normally, and none suffered proteolytic degradation by trypsin. Five variants had normal enzymatic activity, whereas variant p.R29Q was catalytically inactive due to loss of activation by trypsin and variant p.S239C exhibited impaired activity possibly caused by disulfide mispairing. Surprisingly, variant p.G214R had increased activity on a small chromogenic peptide substrate but was markedly defective in cleaving bovine β-casein or the natural CTRC substrates human cationic trypsinogen and procarboxypeptidase A1. Mutation p.G214R is analogous to the evolutionary mutation in human mesotrypsin, which rendered this trypsin isoform resistant to proteinaceous inhibitors and conferred its ability to cleave these inhibitors. Similarly to the mesotrypsin phenotype, CTRC variant p.G214R was inhibited poorly by eglin C, ecotin, or a CTRC-specific variant of SGPI-2, and it readily cleaved the reactive-site peptide bonds in eglin C and ecotin. We conclude that CTRC variants p.R29Q, p.G214R, and p.S239C are risk factors for chronic pancreatitis. Furthermore, the mesotrypsin-like CTRC variant highlights how the same natural mutation in homologous pancreatic serine proteases can evolve a new physiological role or lead to pathology, determined by the biological context of protease function.     

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.     

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。