A quantitative structure-activity relationship study for structurally diverse HIV-1 protease inhibitors: contribution of conformational flexibility to inhibitory activity

In this study, we investigated by linear regression model the SAR data of the 15 HIV-1 protease inhibitors possessing structurally diverse scaffolds. First, a regression model was developed only using the enzyme-inhibitor interaction energy as a term of the model, but did not provide a good correlation with the inhibitory activity (R² = 0.580 and Q² = 0.500). Then, we focused on the conformational flexibility of the inhibitors which may represent the diversity of the inhibitors, and added two conformational parameters into the model, respectively: the number of rotatable bonds of ligands (ΔSrot) and the distortion energy of ligands (ΔElig). The regression model by adding ΔElig successfully improved the quality of the model (R² = 0.771 and Q² = 0.713) while the model with ΔSrot was unsuccessful. The prediction for a training inhibitor by the ΔElig model also showed good agreement with experimental activity. These results suggest that the conformational flexibility of HIV-1 protease inhibitors directly contributes to the enzyme inhibition.     

泛素化结合酶E2抑制剂对草菇15 ℃保鲜的影响

为了改进草菇低温保鲜方法,在15 ℃贮藏条件下,采用泛素化结合酶E2 (UBEV2)抑制剂对草菇子实体进行处理,并开展相关生理指标和基因表达检测。结果表明,使用100 μmol/L的UBEV2抑制剂L345-0044维持了草菇较好的品质,提高了草菇可溶性糖的含量。低温明显提升抑制剂处理下的碱性蛋白酶和中性蛋白酶活力,并证实了低温胁迫显著提高了抑制剂处理下的一种类型蛋白酶肽基赖氨酸金属内肽酶的表达。本研究证实了草菇可溶性糖和高活性的冷诱导金属内肽酶对于延长草菇的低温保鲜时间是必须的。     

Genetically Encoded FRET Biosensor Detects the Enzymatic Activity of Prostate-Specific Antigen

Prostate cancer is the most common cancer among men beyond 50 years old, and ranked the second in mortality. The level of Prostate-specific antigen (PSA) in serum has been a routine biomarker for clinical assessment of the cancer development, which is detected mostly by antibody-based immunoassays. The proteolytic activity of PSA also has important functions. Here a genetically encoded biosensor based on fluorescence resonance energy transfer (FRET) technology was developed to measure PSA activity. In vitro assay showed that the biosensor containing a substrate peptide ‘RLSSYYSGAG’ had 400% FRET change in response to 1 µg/ml PSA within 90 min, and could detect PSA activity at 25 ng/ml. PSA didn’t show enzymatic activity toward the biosensor in serum solution, likely reflecting the existence of other inhibitory factors besides Zn2+. By expressing the biosensor on cell plasma membrane, the FRET responses were significant, but couldn’t distinguish well the cultured prostate cancer cells from non-prostate cancer cells under microscopy imaging, indicating insufficient speci- ficity to PSA. The biosensor with the previously known ‘HSSKLQ’ substrate showed little response to PSA in solution. In summary, we developed a genetically encoded FRET biosensor to detect PSA activity, which may serve as a useful tool for relevant applications, such as screening PSA activation substrates or inhibitors; the purified biosensor protein can also be an alternative choice for measuring PSA activity besides currently commercialized Mu-HSSKLQ-AMC substrate from chemical synthesis.     

Cathepsin-regulated apoptosis

Apoptosis can be mediated by different mechanisms. There is growing evidence that different proteolytic enzymes are involved in the regulation of apoptosis. Cathepsins are proteases which, under physiologic conditions, are localized intralysosomally. In response to certain signals they are released from the lysosomes into the cytoplasm where they trigger apoptotic cell death via various pathways, including the activation of caspases or the release of proapoptotic factors from the mitochondria. Here, we review different mechanisms that induce the release of lysosomal enzymes, and the functional relevance of defined cathepsins in defined models of apoptosis.     

Screening for enzyme activity in turbid suspensions with scattered light

New screening techniques for improved enzyme variants in turbid media are urgently required in many industries such as the detergent and food industry. Here, a new method is presented to measure enzyme activity in different types of substrate suspensions. This method allows a semiquantitative determination of protease activity using native protein substrates. Unlike conventional techniques for measurement of enzyme activity, the BioLector technology enables online monitoring of scattered light intensity and fluorescence signals during the continuous shaking of samples in microtiter plates. The BioLector technique is hereby used to monitor the hydrolysis of an insoluble protein substrate by measuring the decrease of scattered light. The kinetic parameters for the enzyme reaction (V(max,app) and K(m,app)) are determined from the scattered light curves. Moreover, the influence of pH on the protease activity is investigated. The optimal pH value for protease activity was determined to be between pH 8 to 11 and the activities of five subtilisin serine proteases with variations in the amino acid sequence were compared. The presented method enables proteases from genetically modified strains to be easily characterized and compared. Moreover, this method can be applied to other enzyme systems that catalyze various reactions such as cellulose decomposition.     

Examination of the genus Bordetella for specific human IgA protease activity

Abstract Three laboratory strains and 3 clinical isolates of Bordetella pertussis were found to have no IgA protease activity when incubated with radio-labelled IgA. In addition, no IgA protease activity was detected in the Second British Reference Preparation for Pertussis Vaccine, an acellular vaccine produced in Japan, or one strain each of Bordetella parapertussis and Bordetella bronchiseptica .     

Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi

Bacillus subtilis DC33 producing a novel fibrinolytic enzyme was isolated from Ba-bao Douchi, a traditional soybean-fermented food in China. The strong fibrin-specific enzyme subtilisin FS33 was purified to electrophoretic homogeneity using the combination of various chromatographic steps. The optimum temperature, pH value, and pI of subtilisin FS33 were 55°C, 8.0, and 8.7, respectively. The molecular weight was 30 kDa measured by SDS–PAGE under both reducing and non-reducing conditions. The enzyme showed a level of fibrinolytic activity that was about six times higher than that of subtilisin Carlsberg. The first 15 amino acid residues of N-terminal sequence of the enzyme were A-Q-S-V-P-Y-G-I-P-Q-I-K-A-P-A, which are different from that of other known fibrinolytic enzymes. The amidolytic activities of subtilisin FS33 were inhibited completely by 5 mM phenylmethanesulfonyl fluoride (PMSF) and 1 mM soybean trypsin inhibitor (SBTI), but 1,4-dithiothreitol (DTT), β-mercaptoethanol, and p-hydroxymercuribenzoate (PHMB) did not affect the enzyme activity; serine and tryptophan are thus essential in the active site of the enzyme. The highest affinity of subtilisin FS33 was towards N-Succ-Ala-Ala-Pro-Phe-pNA. Therefore, the enzyme was considered to be a subtilisin-like serine protease. The fibrinolytic enzyme had a high degrading activity for the Bβ-chains and Aα-chain of fibrin(ogen), and also acted on thrombotic and fibrinolytic factors of blood, such as plasminogen, urokinase, thrombin, and kallikrein. So subtilisin FS33 was able to degrade fibrin clots in two ways, i.e., (a) by forming active plasmin from plasminogen and (b) by direct fibrinolysis.     

A cuticle-degrading protease (CDEP-1) of Beauveria bassiana enhances virulence

In order to investigate virulence enhancement of entomopathogenic fungi, a Beauveria bassiana-sourced Pr1 protease (CDEP-1) was expressed by a methylotrophic yeast Pichia pastoris and then used as an additive to three gradient sprays of B. bassiana strain (Bb0062) onto apterous green peach aphid Myzus persicae adults in six bioassays. The resultant data fit well to a time–concentration–mortality model. Generally, the LC₅₀ estimates of the fungal pathogen against the aphid species decreased with increasing CDEP-1 concentrations from 0 to 100 µg mL^?1. The LC₅₀s on days 5–7 after spray were reduced by 1.5–2.5-fold at the concentrations of 20–100 µg mL^?1. However, sprays of 20–100 µg CDEP-1 mL^?1 aqueous solution alone had no significant effect on aphid mortality compared to water spray only. Neither did inclusion of inactivated CDEP-1 at a concentration of 50 µg mL^?1 affect significantly the fungal virulence to aphids. Our results confirm for the first time that the cuticle-degrading protease CDEP-1 enhanced fungal virulence due to acceleration of conidial germination and cuticle penetration. This suggests a new approach to utilising the protease in microbial control.