β-Propeller repeats and a PDZ domain in the tricorn protease: predicted self-compartmentalisation and C-terminal polypeptide-binding strategies of substrate selection

Prokaryotic proteases demonstrate a variety of substrate-selection strategies that prevent uncontrolled protein degradation. Proteasomes and ClpXP-like proteases form oligomeric structures that exclude large substrates from central solvated chambers containing their active sites. Monomeric prolyl oligopeptidases have been shown to contain beta-propeller structures that similarly reduce access to their catalytic residues. By contrast, Tsp-like enzymes contain PDZ domains that are thought to specifically target C-terminal polypeptides. We have investigated the sequence of Thermoplasma acidophilum tricorn protease using recently-developed database search methods. The tricorn protease is known to associate into a 20 hexamer capsid enclosing an extremely large cavity that is 37 nm in diameter. It is unknown, however, how this enzyme selects its small oligopeptide substrates. Our results demonstrate the presence in tricorn protease of a PDZ domain and two predicted six-bladed beta-propeller domains. We suggest that the PDZ domain is involved in targeting non-polar C-terminal peptides, similar to those generated by the T. acidophilum proteasome, whereas the beta-propeller domains serve to exclude large substrates from the tricorn protease active site in a similar manner to that previously indicated for prolyl oligopeptidase.     

A screen for immunity genes evolving under positive selection in Drosophila

Genes involved in the immune system tend to have higher rates of adaptive evolution than other genes in the genome, probably because they are coevolving with pathogens. We have screened a sample of Drosophila genes to identify those evolving under positive selection. First, we identified rapidly evolving immunity genes by comparing 140 loci in Drosophila erecta and D. yakuba. Secondly, we resequenced 23 of the fastest evolving genes from the independent species pair D. melanogaster and D. simulans, and identified those under positive selection using a McDonald-Kreitman test. There was strong evidence of adaptive evolution in two serine proteases (persephone and spirit) and a homolog of the Anopheles serpin SRPN6, and weaker evidence in another serine protease and the death domain protein dFADD. These results add to mounting evidence that immune signalling pathway molecules often evolve rapidly, possibly because they are sites of host-parasite coevolution.     

Evidence for positive selection on Drosophila melanogaster seminal fluid protease homologs

Proteins present in the seminal fluid of Drosophila melanogaster (accessory gland proteins Acps) contribute to female postmating behavioral changes, sperm storage, sperm competition, and immunity. Consequently, male-female coevolution and host-pathogen interactions are thought to underlie the rapid, adaptive evolution that characterizes several Acp-encoding genes. We propose that seminal fluid proteases are likely targets of selection due to their demonstrated or potential roles in between-sex interactions and immune processes. We use within- and between-species sequence data for 5 predicted protease-encoding Acp loci to test this hypothesis. Our polymorphism-based analyses find evidence for positive selection at 2 genes, both of which encode predicted serine protease homologs. One of these genes, CG6069, also shows evidence for consistent selection on a subset of codons over a deeper evolutionary time scale. The second gene, CG9997, was previously shown to be essential for normal sperm usage, suggesting that sexual selection may underlie its history of adaptation.     

Screening,selection and development of Bacillus subtilis apr-IBL04 for hyper production of macromolecule alkaline protease

Bacillus subtilis microbe is commonly found in soil and produces proteases on nitrogen and carbon-containing sources and increases the fertility rate by degrading nitrogenous organic materials. The present study was aimed to develop hyper producing mutant strain of B. subtilis for the production of proteases, to improve the process variables by the response surface methodology (RSM) under central composite design (CCD) and the production of protease by the particular mutant strain in a liquid state fermentation media. The mutation of the strain was carried out using ethidium bromide. Pure B. subtilis strain was collected and screened for hyper-production of protease. The production of protease by mutant B. subtilis strain was optimized by varying temperature, inoculum size, pH and incubation time under liquid state fermentation. The CCD model were found to be reliable with r² of 0.999. The maximum enzyme activity of B. subtilis IBL-04 mutant with 3 mL/100 mL inoculum size, 72 h fermentation time, pH 8, and 45 °C temperature was developed with enzyme activity 631.09 U/mL, indicates 1–7-fold increase in enzyme activity than the parent strain having 82.32 U/mL activity. These characteristics render its potential use in industries for pharmaceutical and dairy formulation.     

Navigation inside a protease: substrate selection and product exit in the tricorn protease from Thermoplasma acidophilum

Chaperonins are multi-subunit double-ring complexes that mediate the folding of nascent or denatured proteins. Gene duplication has been a potent force in the evolution of chaperonins in Archaea. Here we show that gene conversion has also been an important factor. We utilized a novel maximum likehood-based phylogenetic method for scanning DNA sequence alignments for regions of anomalous phylogenetic signal, such as those affected by gene conversion. Our results suggest that in crenarchaeotes, where an ancient gene duplication producing alpha and beta subunits took place in the common ancestor of the Pyrodictium, Aeropyrum, Pyrobaculum and Sulfolobus lineages, multiple independent gene conversions have occurred between the alpha and beta genes independently in each of these groups. Significantly, the conversions have repeatedly homogenized the region of the gene encoding the substrate-binding domain. This suggests that while the alpha and beta subunits in crenarchaeotes share only 50-60% overall amino acid sequence identity, they do not possess distinct roles in the binding of substrate. Cryptic gene conversion between distantly related paralogs may be more common than is currently appreciated, and could be a significant factor in slowing the functional differentiation of proteins encoded by duplicate genes long after their duplication.     

Control of xylanase production without protease activity in Bacillus sp. by selection of nitrogen source

Maximum xylanase activity, of 380 IU ml^–1, with negligible protease activity, occurred when Bacillus SSP-34 was grown for 96 h with yeast extract and peptone each at 0.25%. Other concentrations of the combination gave xylanase activities less than 66% of that with the optimum nitrogen source concentration and protease activities in the range of 0.01–0.045 IU ml^–1.     

基于二层特征筛选的HIV-1蛋白酶特异位点预测

在抗艾滋病治疗中,HIV-1蛋白酶抑制剂发挥着重要作用。对于HIV-1蛋白酶裂解作用位点的研究有助于找到新的治疗靶点。为了对HIV-1蛋白酶特异位点进行预测,本研究用氨基酸索引数据库(Amino Acid Index,AAIndex)中的531个氨基酸物理化学性质参数直接表征肽样本的结构,通过二层特征筛选,最终将4248个表征参数降为57个表征参数。分别采取四种核函数进行HIV-1蛋白酶特异位点的支持向量机(SVM)建模,并通过10折交叉验证及外部测试集方法来验证建模的准确性。结果表明选取NormalizePolyKernel核函数进行SVM建模效果优于其他核函数(PolyKernel、PUK、RBFKernel),所建立的模型对于训练集的10组交叉验证预测准确率达到93.947%,对于外部测试集的预测正确率达到93.684%。     

Activation of the West Nile virus NS3 protease: Molecular dynamics evidence for a conformational selection mechanism

The flaviviral nonstructural 3 protease (NS3pro) is essential for virus replication and is therefore a pharmaceutically relevant target to fight Dengue and West Nile virus (WNV). NS3pro is a chymotrypsin‐like serine protease which requires a polypeptide cofactor (NS2B) for activation. Recent X‐ray crystallography studies have led to the suggestion that the substrate binds to the two‐component NS2B‐NS3pro enzyme by an induced‐fit mechanism. Here, multiple explicit water molecular dynamics simulations of the WNV NS2B‐NS3pro enzyme show that the active conformation of the NS2B cofactor (in which its β‐loop is part of the substrate binding site) is stable over a 50‐ns time scale even in the absence of the inhibitor. The partial and reversible opening of the NSB2 β‐loop and its correlated motion with an adjacent NS3pro loop, both observed in the simulations started from the active conformation, are likely to facilitate substrate binding and product release. Moreover, in five of eight simulations without inhibitor (started from two X‐ray structures both with improperly formed oxyanion hole) the Thr132‐Gly133 peptide bond flips spontaneously thereby promoting the formation of the catalytically competent oxyanion hole, which then stays stable until the end of the runs. The simulation results provide evidence at atomic level of detail that the substrate binds to the NS2B‐NS3pro enzyme by conformational selection, rather than induced‐fit mechanism.     

Rapid selection of potyviral cross-protection effective mutants from the local lesion host after nitrous acid mutagenesis

Zucchini yellow mosaic virus (ZYMV) seriously damages cucurbits worldwide. Control of ZYMV by cross-protection has been practised for decades, but selecting useful mild viruses is time-consuming and laborious. Most attenuated potyviruses used for cross-protection do not induce hypersensitive reaction (HR) in Chenopodium quinoa, a local lesion host Chenopodium quinoa. Here, severe ZYMV TW-TN3 tagged with green fluorescent protein (GFP), designated ZG, was used for nitrous acid mutagenesis. From three trials, 11 mutants were identified from fluorescent spots without HR in inoculated C. quinoa leaves. Five mutants caused attenuated symptoms in squash plants. The genomic sequences of these five mutants revealed that most of the nonsynonymous changes were located in the HC-Pro gene. The replacement of individual mutated HC-Pros in the ZG backbone and an RNA silencing suppression (RSS) assay indicated that each mutated HC-Pro is defective in RSS function and responsible for reduced virulence. Four mutants provided high degrees of protection (84%–100%) against severe virus TW-TN3 in zucchini squash plants, with ZG 4-10 being selected for removal of the GFP tag. After removal of the GFP gene, Z 4-10 induced symptoms similar to ZG 4-10 and still provided 100% protection against TW-TN3 in squash, thus is considered not a genetically engineered mutant. Therefore, using a GFP reporter to select non-HR mutants of ZYMV from C. quinoa leaves is an efficient way to obtain beneficial mild viruses for cross-protection. This novel approach is being applied to other potyviruses.     

几种离子及有机物对蜡样芽胞杆菌蛋白酶活力的影响

研究不同浓度的金属离子及有机物对蜡样芽胞杆菌蛋白酶活力的影响,结果表明①五种无机离子中Mg     

PROFICS: A bacterial selection system for directed evolution of proteases

Proteases serve as important tools in biotechnology and as valuable drugs or drug targets. Efficient protein engineering methods to study and modulate protease properties are thus of great interest for a plethora of applications. We established PROFICS (PRotease Optimization via Fusion-Inhibited Carbamoyltransferase-based Selection), a bacterial selection system, which enables the optimization of proteases for biotechnology, therapeutics or diagnosis in a simple overnight process. During the PROFICS process, proteases are selected for their ability to specifically cut a tag from a reporter enzyme and leave a native N-terminus. Precise and efficient cleavage after the recognition sequence reverses the phenotype of an Escherichia coli knockout strain deficient in an essential enzyme of pyrimidine synthesis. A toolbox was generated to select for proteases with different preferences for P1′ residues (the residue immediately following the cleavage site). The functionality of PROFICS is demonstrated with viral proteases and human caspase-2. PROFICS improved caspase-2 activity up to 25-fold after only one round of mutation and selection. Additionally, we found a significantly improved tolerance for all P1′ residues caused by a mutation in a substrate interaction site. We showed that this improved activity enables cells containing the new variant to outgrow cells containing all other mutants, facilitating its straightforward selection. Apart from optimizing enzymatic activity and P1′ tolerance, PROFICS can be used to reprogram specificities, erase off-target activity, optimize expression via tags/codon usage, or even to screen for potential drug-resistance-conferring mutations in therapeutic targets such as viral proteases in an unbiased manner.     

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.     

Recent progress in the discovery of inhibitors targeting coronavirus proteases

Coronaviruses (CoVs) can cause highly prevalent diseases in humans and animals. The fatal outbreak of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) highlights the threat posed by this unique virus subfamily. However, no specific drugs have been approved to treat CoV-associated diseases to date. The CoV proteases, which play pivotal roles in viral gene expression and replication through a highly complex cascade involving the proteolytic processing of replicase polyproteins, are attractive targets for drug design. This review summarizes the recent advances in biological and structural studies, together with the development of inhibitors targeting CoV proteases, particularly main proteases (M^pros), which could help develop effective treatments to prevent CoV infection.

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Purification and characterization of extracellular alkaline serine protease from Stenotrophomonas maltophilia strain S-1

AIMS: The present study was conducted by screening soil bacteria in an attempt to isolate a bacterium that produced extracellular alkaline protease, and for purification and characterization of the protease. METHODS AND RESULTS: Soil bacteria were screened by growth on casein as the sole carbon source. Characterization of a strain isolated from soil of Abashiri, Japan indicated a taxonomic affiliation to Stenotrophomonas maltophilia, and was named S-1 strain. The purified S-1 protease, designed S. maltophilia Protease-1 (SmP-1), exhibited an optimal pH of 12.0, optimal reaction temperature of 50 degrees C and a molecular mass of approximately 40 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The cleavage sites of the oxidized-insulin B chain by SmP-1 were identified as Leu6-Cys7, Cys7-Gly8, Tyr16-Leu17 and Leu17-Val18. The N-terminal amino acid sequence of the purified alkaline protease was determined as NH2-SASAPMVSGVAALVLE. CONCLUSION: A novel extracellular alkaline serine protease was isolated from S. maltophilia strain S-1. The optimal pH of the proteolytic activity was pH 12.0. SIGNIFICANCE AND IMPACT OF THE STUDY: The extremely high optimal pH and heat stability of the alkaline serine protease SmP-1 might make it widely applicable to food and other industries.     

Protein aggregation in Escherichia coli: role of proteases

Protein aggregation is involved in several human diseases, and presumed to be an important process in protein quality control. In bacteria, aggregation of proteins occurs during stress conditions, such as heat shock. We studied the protein aggregates of Escherichia coli during heat shock. Our results demonstrate that the concentration and diversity of proteins in the aggregates depend on the availability of proteases. Aggregates obtained from mutants in the Lon (La) protease contain three times more protein than wild-type aggregates and show the broadest protein diversity. The results support the assumption that protein aggregates are formed from partially unfolded proteins that were not refolded by chaperones or degraded by proteases.     

Streptococcus pneumoniae IgA1 protease: A metalloprotease that can catalyze in a split manner in vitro

IgA1 proteases (IgA1P) from diverse pathogenic bacteria specifically cleave human immunoglobulin A1 (IgA1) at the hinge region, thereby thwarting protective host immune responses. Streptococcus pneumoniae (S. pneumoniae) IgA1P shares no sequence conservation with serine or cysteine types of IgA1Ps or other known proteins, other than a conserved HExxH Zn‐binding motif (1604‐1608) found in metalloproteases. We have developed a novel expression system to produce the mature S. pneumoniae IgA1P and we have discovered that this form is both attached to the bacterial cell surface and released in its full form. Our data demonstrate that the S. pneumoniae IgA1P comprises two distinct regions that associate to form an active metalloprotease, the first such example of a metalloprotease that can be split in vitro and recombined to form an active enzyme. By capitalizing on this novel domain architecture, we show that the N‐terminal region of S. pneumoniae IgA1P comprises the primary binding region for IgA1, although the C‐terminal region of S. pneumoniae IgA1P is necessary for cleavage of IgA1. Our findings lend insight into the protein domain architecture of the S. pneumoniae IgA1P and function of this important virulence factor for S. pneumoniae infection.     

Positive selection of digestive proteases in Daphnia: A mechanism for local adaptation to cyanobacterial protease inhibitors

Due to the combined effects of global warming and eutrophication, the frequency of deleterious cyanobacterial blooms in freshwater ecosystems has increased. In line with this, local adaptation of the aquatic keystone herbivore Daphnia to cyanobacteria has received major attention. Besides microcystins, the most frequent cyanobacterial secondary metabolites in such blooms are protease inhibitors (PIs). Recently, it has been shown that a protease gene showed copy number variation between four D. magna populations that differed in tolerance to PIs. From that study, we chose two distinct populations of D. magna which had or had not coexisted with cyanobacteria in the past. By calculating F_ST values, we found that the two populations were genetically more distant in the protease loci than in neutral loci. Population genetic tests applied to the tolerant population revealed that positive selection was most probably acting on the gene loci of the digestive protease CT448 and CT802. We conclude that the selection of digestive proteases and subsequent reduction in copy number is the molecular basis of evolutionary changes leading to local adaptation to PIs.     

Purification and characterization of an alkaline protease from Pseudomonas aeruginosa MN1

An alkaline protease produced by Pseudomonas aeruginosa MN1, isolated from an alkaline tannery waste water, was purified and characterized. The enzyme was purified 25-fold by gel filtration and ion exchange chromatography to a specific activity of 82350 U mg⁻¹. The molecular weight of the enzyme was estimated to be 32000 daltons. The optimum pH and temperature for the proteolytic activity were pH 8.00 and 60°C, respectively. Enzyme activity was inhibited by EDTA suggesting that the preparation contains a metalloprotease. Enzyme activity was strongly inhibited by Zn²⁺, Cu²⁺ and Hg²⁺(5 mM), while Ca²⁺ and Mn²⁺ resulted in partial inhibition. The enzyme is different from other Pseudomonas aeruginosa alkaline proteases in its stability at high temperature; it retained more than 90% and 66% of the initial activity after 15 and 120 min incubation at 60°C. Journal of Industrial Microbiology & Biotechnology (2000) 24, 291–295. Received 09 June 1999/ Accepted in revised form 24 January 2000     

Computational selection of flavonoid compounds as inhibitors against SARS-CoV-2 main protease,RNA-dependent RNA polymerase and spike proteins: A molecular docking study

An outbreak of Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has been recognized as a global health concern. Since, no specific antiviral drug is proven effective for treatment against COVID-19, identification of new therapeutics is an urgent need. In this study, flavonoid compounds were analyzed for its inhibitory potential against important protein targets of SARS-CoV-2 using computational approaches. Virtual docking was performed for screening of flavonoid compounds retrieved from PubChem against the main protease of SARS-CoV-2 using COVID-19 docking server. The cut off of dock score was set to >?9 kcal/mol and screened compounds were individually docked against main protease, RNA-dependent RNA polymerase, and spike proteins using AutoDock 4.1 software. Finally, lead flavonoid compounds were subjected to ADMET analysis. A total of 458 flavonoid compounds were virtually screened against main protease target and 36 compounds were selected based on the interaction energy value >?9 kcal/mol. Furthermore, these compounds were individually docked against protein targets and top 10 lead compounds were identified. Among the lead compounds, agathisflavone showed highest binding energy value of ?8.4 kcal/mol against main protease, Albireodelphin showed highest dock score of ?9.8 kcal/mol and ?11.2 kcal/mol against RdRp, and spike proteins, respectively. Based on the high dock score and ADMET properties, top 5 lead molecules such as Albireodelphin, Apigenin 7-(6″-malonylglucoside), Cyanidin-3-(p-coumaroyl)-rutinoside-5-glucoside, Delphinidin 3-O-beta-D-glucoside 5-O-(6-coumaroyl-beta-D-glucoside) and (-)-Maackiain-3-O-glucosyl-6″-O-malonate were identified as potent inhibitors against main protease, RdRp, and spike protein targets of SARS-CoV-2. These all compounds are having non-carcinogenic and non-mutagenic properties. This study finding suggests that the screened compounds include Albireodelphin, Apigenin 7-(6″-malonylglucoside), Cyanidin-3-(p-coumaroyl)-rutinoside-5-glucoside, Delphinidin 3-O-beta-D-glucoside 5-O-(6-coumaroyl-beta-D-glucoside) and (-)-Maackiain-3-O-glucosyl-6″-O-malonate could be the potent inhibitors of SARS-CoV-2 targets.