Interaction of Proteinaceous Protease Inhibitor of Bacillus subtilis with Intracellular Proteases from the Same Strain

Intracellular protease inhibitor of B. subtilis IFO 3027 inhibits cytoplasmic serine proteases (IP-I, IP-II and IP-III), but it does not affect membrane-bound serine protease from the same strain. Inhibitory equivalent and binding titration studies revealed that the inhibitor binds and inhibits one mol of IP-I (a major protease in the cells). The inhibitor constant, Ki, against IP-I is estimated to be approximately 10^–9 m. The inhibitor binds and inhibits IP-II and IP-III in the same manner as IP-I, but its association with these enzymes is strongly repressed by the substrate (casein). Incubation of the inhibitor with membrane-bound protease results in the loss of the inhibitory activity.     

Skeletal Muscle-specific Calpain Is an Intracellular Na+-dependent Protease

Because intracellular [Na⁺] is kept low by Na⁺/K⁺-ATPase, Na⁺ dependence is generally considered a property of extracellular enzymes. However, we found that p94/calpain 3, a skeletal-muscle-specific member of the Ca²⁺-activated intracellular “modulator proteases” that is responsible for a limb-girdle muscular dystrophy (“calpainopathy”), underwent Na⁺-dependent, but not Cs⁺-dependent, autolysis in the absence of Ca²⁺. Furthermore, Na⁺ and Ca²⁺ complementarily activated autolysis of p94 at physiological concentrations. By blocking Na⁺/K⁺-ATPase, we confirmed intracellular autolysis of p94 in cultured cells. This was further confirmed using inactive p94:C129S knock-in (p94CS-KI) mice as negative controls. Mutagenesis studies showed that much of the p94 molecule contributed to its Na⁺/Ca²⁺-dependent autolysis, which is consistent with the scattered location of calpainopathy-associated mutations, and that a conserved Ca²⁺-binding sequence in the protease acted as a Na⁺ sensor. Proteomic analyses using Cs⁺/Mg²⁺ and p94CS-KI mice as negative controls revealed that Na⁺ and Ca²⁺ direct p94 to proteolyze different substrates. We propose three roles for Na⁺ dependence of p94; 1) to increase sensitivity of p94 to changes in physiological [Ca²⁺], 2) to regulate substrate specificity of p94, and 3) to regulate contribution of p94 as a structural component in muscle cells. Finally, this is the first example of an intracellular Na⁺-dependent enzyme.     

Species-Specific Serological Detection for Schistosomiasis by Serine Protease Inhibitor (SERPIN) in Multiplex Assay

Background

Both Schistosoma mansoni and Schistosoma haematobium cause schistosomiasis in sub-Saharan Africa. We assessed the diagnostic value of selected Schistosoma antigens for the development of a multiplex serological immunoassay for sero-epidemiological surveillance.

Methodology/Principal Findings

Diagnostic ability of recombinant antigens from S. mansoni and S. haematobium was assessed by Luminex multiplex immunoassay using plasma from school children in two areas of Kenya, endemic for different species of schistosomiasis. S. mansoni serine protease inhibitor (SERPIN) and Sm-RP26 showed significantly higher reactivity to patient plasma as compared to the control group. Sm-Filamin, Sm-GAPDH, Sm-GST, Sm-LAP1, Sm-LAP2, Sm-Sm31, Sm-Sm32 and Sm-Tropomyosin did not show difference in reactivity between S. mansoni infected and uninfected pupils. Sm-RP26 was cross-reactive to plasma from S. haematobium patients, whereas Sm-SERPIN was species-specific. Sh-SEPRIN was partially cross-reactive to S. mansoni infected patients. ROC analysis for Sm-RP26, Sm-SERPIN and Sh-SERPIN showed AUC values of 0.833, 0.888 and 0.947, respectively. Using Spearman’s rank correlation coefficient analysis, we also found significant positive correlation between the number of excreted eggs and median fluorescence intensity (MFI) from the multiplex immunoassays for Sm-SERPIN (ρ = 0.430, p-value = 0.003) and Sh-SERPIN (ρ = 0.433, p-value = 0.006).

Conclusions/Significance

Sm-SERPIN is a promising species-specific diagnostic antigen. Sh-SEPRIN was partially cross-reactive to S. mansoni infected patients. SERPINs showed correlation with the number of excreted eggs. These indicate prospects for inclusion of SERPINs in the multiplex serological immunoassay system.     

A Protease Inhibitor from Penicillium cyclopium

When the protease inhibitor from Penicillium cyclopium was mixed with the acid protease of the mold at acid pH formed a precipitate consisting of a enzyme-inhibitor complex. The precipitation occurred maximally at pH 3.0 and was interfered with by increasing amounts of salts and other protein. Subsequent incubation of the complex brought about inactivation of the enzyme and the inactivation was found to be accompanied by modification of the enzyme so that less was precipitable with trichloroacetic acid. Paper chromatography revealed that the enzyme on complete inactivation had been degraded to several fragments or polypeptides. The inhibitor acted on the enzyme in a catalytic fashion, bringing about degradation of more than a stoichiometric amount of enzyme. The proposed mechanism of the inhibitor action involved acceleration of auto-digestion of the enzyme which splits the molecule into small fragments and abolishes the activity.     

A Protease Inhibitor from Penicillium cyclopium

A protease inhibitor produced by Penicillium cyclopium on solid cultures of wheat bran was purified by means of column chromatography on Duolite A-2 and DEAE-cellulose, acetone precipitation and lyophilization. The purified inhibitor obtained as a white, floccose and hygroscopic substance was monodisperse by ultracentrifugal analysis. It was found to be an acidic macro-molecule of a molecular weight of about 5000. The chemical analyses rejected the possibility of the presence of amino acids, peptides, sugars, amino sugars, or uronic acids in the inhibitor molecule.

Properties of a protease inhibitor from Penicillium cyclopium were studied. The pH range of the inhibitor action is restricted to acid pH, optimally at pH 3. Increasing temperature accelerates its action upon enzyme. The inhibitor causes enzyme inactivation in proportion to its concentration. It is fairly stable in an acid solution but unstable in an alkaline solution. It undergoes destruction by heat, hydrogen peroxide and ascorbic acid. The inhibitor reversibly combines with Al³⁺, Fe³⁺, Ag⁺ and Cu²⁺ to produce a precipitate. Salts interfer with the inhibitor activity. Generally, acid proteases from various penicillia are susceptible to the inhibitor while those from other genera are resistant.     

Isolation of Alkaline Protease Inhibitor Producing Microorganisms

For the purpose of obtaining microorganisms capable of producing alkaline protease inhibitor, screening test was carried out. One strain of microorganisms (No. S–3253) showed strong ability to produce alkaline protease inhibitor.

The morphological and physiological characteristics of strain S–3253 were studied. This strain was found to belong to Streptomyces albogriseolus.

When the strain S–3253 was cultivated at 45°C with a medium containing 4% polypepton, 2% starch, 0.1% yeast extract, 0.1% NaCl, 0.1% K₂HPO₄ and 0.05% MgSO₄-7H₂O in shake-flasks (pH 7.0), the highest activity was obtained after 40~48 hr cultivation.     

Molecular Phylogenetic Characterization of Streptomyces Protease Inhibitor Family

We previously found that proteinaceous protease inhibitors homologous to Streptomyces subtilisin inhibitor (SSI) are widely produced by various Streptomyces species, and we designated them ``SSI-like proteins' (Taguchi S, Kikuchi H, Suzuki M, Kojima S, Terabe M, Miura K, Nakase T, Momose H [1993] Appl Environ Microbiol 59:4338–4341). In this study, SSI-like proteins from five strains of the genus Streptoverticillium were purified and sequenced, and molecular phylogenetic trees were constructed on the basis of the determined amino acid sequences together with those determined previously for Streptomyces species. The phylogenetic trees showed that SSI-like proteins from Streptoverticillium species are phylogenetically included in Streptomyces SSI-like proteins but form a monophyletic group as a distinct lineage within the Streptomyces proteins. This provides an alternative phylogenetic framework to the previous one based on partial small ribosomal RNA sequences, and it may indicate that the phylogenetic affiliation of the genus Streptoverticillium should be revised. The phylogenetic trees also suggested that SSI-like proteins possessing arginine or methionine at the P1 site, the major reactive center site toward target proteases, arose multiple times on independent lineages from ancestral proteins possessing lysine at the P1 site. Most of the codon changes at the P1 site inferred to have occurred during the evolution of SSI-like proteins are consistent with those inferred from the extremely high G + C content of Streptomyces genomes. The inferred minimum number of amino acid replacements at the P1 site was nearly equal to the average number for all the variable sites. It thus appears that positive Darwinian selection, which has been postulated to account for accelerated rates of amino acid replacement at the major reaction center site of mammalian protease inhibitors, may not have dictated the evolution of the bacterial SSI-like proteins. Received: 23 August 1996 / Accepted: 20 November 1996     

Correlation of Epididymal Protease Inhibitor and Fibronectin in Human Semen

Objective

Epididymal protease inhibitor (Eppin) was located on the surface of spermatozoa and modulates the liquefaction of human semen. Here, we identify the correlative protein partner of Eppin to explore the molecular mechanism of liquefaction of human semen.

Methods

(1) Human seminal vesicle proteins were transferred on the membrane by Western blotting and separated by 2-D electrophoresis and incubated in recombinant Eppin. The correlative protein was identified by Mass Spectrometry (MS) (2). Western blotting was used to determine the relation of rEppin and rFibronectin(Fn); (3) Co-localization in spermatozoa were detected using immunofluorescence; (4) Correalation of Eppin and Fn was proved by co-immunoprecipitation.

Results

Fn was identified as the binding partner of recombinant Eppin by MS. Recombinant of Eppin was made and demonstrated that the Eppin fragment binds the fn 607-1265 fragment. The Eppin-Fn complex presents on the sperm tail and particularly in the midpiece region of human ejaculated spermatozoa. Immunoprecipitation indicated that Eppin in the spermatozoa lysates was complexed with Fn.

Conclusions

Our study demonstrates that Eppin and Fn bind to each other in human semen and on human ejaculated spermatozoa. Eppin-Fn complex may involve in semen coagulation, liquefaction and the survival and preparation of spermatozoa for fertility in the female reproductive tract.     

蛋白酶抑制剂抗虫基因工程研究进展

综述了蛋白酶抑制剂分类、作用机理、自身优势以及研究进展。根据当前的研究现状、存在问题 ,提出了解决措施 ,并对蛋白酶抑制剂的研究前景进行了展望     

马铃薯蛋白酶A抑制剂的纯化与性质

采用硫酸铵沉淀、Sephadex G-50和CM柱层析,从马铃薯汁中纯化蛋白酶A（protease A,PrA）抑制剂,并对其部分性质进行研究。蛋白酶A抑制剂为单亚基,相对分子质量为16．71kDa。热稳定性良好,抑制最佳pH范围为5—5．5,最佳反应时间为1．5h,对PrA抑制类型为竞争和非竞争性混合抑制作用。     

Protease and Protease Inhibitor Activity in Rat Skeletal Muscle during Growth,Protein Deficiency and Fasting

Growing rats fed a protein deficient diet showed an increase in acid and alkaline protease activity and a decrease in protease inhibitor activity. Similar results were found after two and four day fasting periods. Animals on diets of 10% and 20% casein showed parallel protease and protease inhibitor activity when compared to each other at similar body weight periods.     

甲烷古菌群感效应信号分子的检测

竹节状甲烷鬃菌(Methanosaeta harundinacea)6Ac是本实验室分离自厌氧颗粒污泥中的甲烷古菌新种。该菌具有短杆(3μm-5μm)和长链状(>200μm)两种细胞形态,且与细胞密度相关,暗示该菌可能存在群感效应调控的细胞形态变化。【目的】验证该菌存在群感效应信号分子并与细胞形态变化相关。【方法】用高丝氨酸内酯指示菌Agrobacterium tumefaciens NTL4检测菌株6Ac的培养液,并用购买的高丝氨酸内酯标准品加入短杆菌株6Ac检测形态变化。【结果】菌株6Ac的培养液中含有高丝氨酸内酯类物质。实验证明化学合成的高丝氨酸内酯N-(β-酮基)辛酰高丝氨酸内酯能够促进竹节状甲烷鬃菌的长链细胞形成。而且在马氏甲烷八叠球菌(Methanosarcina mazei)、热自养甲烷杆菌(Methanothermobacter thermautotrophicus)和甲酸甲烷杆菌(Methanobacterium formicicum)的培养液中也检测到了高丝氨酸内酯。【结论】多种甲烷古菌可以产生高丝氨酸内酯类物质,并可能以此类物质作为群感效应的信号分子。     

Location of a Protease and its Inhibitor in the Barley Kernel

Tissues of barley caryopsis and seedling were examined for the protease, BAPAase, and an inhibitor. The enzyme was present in extracts of alevn-one but was absent from aleurone incubation media and extracts of: embryo with scutellum; seedling with scutellum and rootlets, and endosperm that was free of aleurone tissue. The enzyme was present in non-incubated aleurone and did not increase significantly during incubation under conditions where alpha-amylase increased in the medium and tissue. Addition of gibberellie acid produced no detectable increase in BAPAase. Extracts of endosperm had weak BAPAase-inhibitory activity; embryo or seedling extracts produced strong inhibition. The inhibitor present in these extracts was dialyzable.     

Studies on a Doubleheaded Protease Inhibitor from Phaseolus mungo

A doubleheaded protease inhibitor showing inhibition of bovine pancreatic trypsin and α-chymotrypsin was isolated and purified from the seeds of Phaseolus mungo. The molecular weight of the protease inhibitor was found to be 14.2 kD by SDS-PAGE analysis and gel filtration. The native inhibitor inhibited trypsin and α-chymotrypsin stoichiometrically at the molar ratio 1:1 and 2:1 respectively. The Ki app for trypsin was found to be 0.35 nM and for α-chymotrypsin to be 2.4 nM. Bovine pepsin was not inhibited by the inhibitor. However, the pepsin treated inhibitor was still able to inhibit trypsin and α-chymotrypsin. The inhibitor was stable in 8M urea. Addition of 0.2 M mercaptoethanol resulted in significant loss of inhibitory activity. The inhibitor was extremely heat stable with only 50% loss of inhibitory activity after heating for 100°C for 20 min. Thus, the Phaseolus mungo trypsin/chymotrypsin inhibitor resembles other Bowman-Birk protease inhibitors.     

Deep Sequencing of Protease Inhibitor Resistant HIV Patient Isolates Reveals Patterns of Correlated Mutations in Gag and Protease

While the role of drug resistance mutations in HIV protease has been studied comprehensively, mutations in its substrate, Gag, have not been extensively cataloged. Using deep sequencing, we analyzed a unique collection of longitudinal viral samples from 93 patients who have been treated with therapies containing protease inhibitors (PIs). Due to the high sequence coverage within each sample, the frequencies of mutations at individual positions were calculated with high precision. We used this information to characterize the variability in the Gag polyprotein and its effects on PI-therapy outcomes. To examine covariation of mutations between two different sites using deep sequencing data, we developed an approach to estimate the tight bounds on the two-site bivariate probabilities in each viral sample, and the mutual information between pairs of positions based on all the bounds. Utilizing the new methodology we found that mutations in the matrix and p6 proteins contribute to continued therapy failure and have a major role in the network of strongly correlated mutations in the Gag polyprotein, as well as between Gag and protease. Although covariation is not direct evidence of structural propensities, we found the strongest correlations between residues on capsid and matrix of the same Gag protein were often due to structural proximity. This suggests that some of the strongest inter-protein Gag correlations are the result of structural proximity. Moreover, the strong covariation between residues in matrix and capsid at the N-terminus with p1 and p6 at the C-terminus is consistent with residue-residue contacts between these proteins at some point in the viral life cycle.     

生长抑素联合抑肽酶治疗急性重症胰腺炎临床疗效观察

目的:观察生长抑素联合抑肽酶治疗急性重症胰腺炎的临床疗效.方法:将急性重症胰腺炎患者48例随机分为两组:A组和B组,A组患者在常规治疗基础上加用生长抑素联合抑肤酶治疗,B组患者在常规治疗基础上加用生长抑素.比较两组患者的腹痛、腹胀缓解时间、血淀粉酶恢复正常时间、WBC恢复正常时间以及感染率、痛死率、并发症发生率、住院时间和住院费用.结果:A组患者腹痛、腹胀缓解时间、血淀粉酶恢复正常时间、WBC恢复正常时间较B组明显缩短,感染率、病死率、并发症发生率、住院时间及住院费用较B组明显降低.结论:生长抑素联合抑肽酶治疗急性重症胰腺炎效果明显,可较早缓解症状并缩短病程、降低感染率、病死率、并发症发生率从而缩短住院时间,降低住院费用.     

Identification of Lympho-Epithelial Kazal-Type Inhibitor 2 in Human Skin as a Kallikrein-Related Peptidase 5-Specific Protease Inhibitor

Kallikreins-related peptidases (KLKs) are serine proteases and have been implicated in the desquamation process of the skin. Their activity is tightly controlled by epidermal protease inhibitors like the lympho-epithelial Kazal-type inhibitor (LEKTI). Defects of the LEKTI-encoding gene serine protease inhibitor Kazal type (Spink)5 lead to the absence of LEKTI and result in the genodermatose Netherton syndrome, which mimics the common skin disease atopic dermatitis. Since many KLKs are expressed in human skin with KLK5 being considered as one of the most important KLKs in skin desquamation, we proposed that more inhibitors are present in human skin. Herein, we purified from human stratum corneum by HPLC techniques a new KLK5-inhibiting peptide encoded by a member of the Spink family, designated as Spink9 located on chromosome 5p33.1. This peptide is highly homologous to LEKTI and was termed LEKTI-2. Recombinant LEKTI-2 inhibited KLK5 but not KLK7, 14 or other serine proteases tested including trypsin, plasmin and thrombin. Spink9 mRNA expression was detected in human skin samples and in cultured keratinocytes. LEKTI-2 immune-expression was focally localized at the stratum granulosum and stratum corneum at palmar and plantar sites in close localization to KLK5. At sites of plantar hyperkeratosis, LEKTI-2 expression was increased. We suggest that LEKTI-2 contributes to the regulation of the desquamation process in human skin by specifically inhibiting KLK5.     

Effectiveness of Ritonavir-Boosted Protease Inhibitor Monotherapy in Clinical Practice Even with Previous Virological Failures to Protease Inhibitor-Based Regimens

Background and Objective

Significant controversy still exists about ritonavir-boosted protease inhibitor monotherapy (mtPI/rtv) as a simplification strategy that is used up to now to treat patients that have not experienced previous virological failure (VF) while on protease inhibitor (PI) -based regimens. We have evaluated the effectiveness of two mtPI/rtv regimens in an actual clinical practice setting, including patients that had experienced previous VF with PI-based regimens.

Methods

This retrospective study analyzed 1060 HIV-infected patients with undetectable viremia that were switched to lopinavir/ritonavir or darunavir/ritonavir monotherapy. In cases in which the patient had previously experienced VF while on a PI-based regimen, the lack of major HIV protease resistance mutations to lopinavir or darunavir, respectively, was mandatory. The primary endpoint of this study was the percentage of participants with virological suppression after 96 weeks according to intention-to-treat analysis (non-complete/missing = failure).

Results

A total of 1060 patients were analyzed, including 205 with previous VF while on PI-based regimens, 90 of whom were on complex therapies due to extensive resistance. The rates of treatment effectiveness (intention-to-treat analysis) and virological efficacy (on-treatment analysis) at week 96 were 79.3% (CI₉₅, 76.8−81.8) and 91.5% (CI₉₅, 89.6–93.4), respectively. No relationships were found between VF and earlier VF while on PI-based regimens, the presence of major or minor protease resistance mutations, the previous time on viral suppression, CD4⁺ T-cell nadir, and HCV-coinfection. Genotypic resistance tests were available in 49 out of the 74 patients with VFs and only four patients presented new major protease resistance mutations.

Conclusion

Switching to mtPI/rtv achieves sustained virological control in most patients, even in those with previous VF on PI-based regimens as long as no major resistance mutations are present for the administered drug.     

Isolation and Crystallization of Microbial Alkaline Protease Inhibitor,S-SI

S–SI, an microbial alkaline protease inhibitor, was produced in the culture broth of Streptomyces albogriseolus S–3253. S–SI was isolated from culture filtrate by salting out with ammonium sulfate, column chromatographies on DEAE-cellulose and Sephadex G–100. S–SI was also isolated by pH adjustment of the effluent from DEAE-cellulose column.

Crystallization of S–SI was carried out, and two distinct shaped crystals, needle shaped and rhombic crystal, were obtained. Homogeneity of these crystals were identified by polyacrylamide disc electrophoresis at various pHs.

S–SI retained its perfect inhibitory activity after treatment at 37°C for 25 hr in the pH range from 3 to 10, and 100°C for 10 min in the pH range from 4 to 6.

From the examination of molecular weight determination with Sephadex G–100, the molecular weight of S–SI was decided to be about 27,000.

S–SI was found to be highly specific towards microbial alkaline proteases.