Peptide Bond Hydrolysis Research Articles

Importance of being cross-linked for the bacterial cell wall.

The bacterial cell wall is primarily composed of a mesh of glycan strands cross-linked by peptide bridges and is essential for safeguarding the cell. The structure of the cell wall has to be stiff enough to bear the high turgor pressure and sufficiently tough to ensure protection against failure. Here we explore the role of various design features of the cell in enhancing the toughness of the cell wall. We explain how the glycan strand length distribution, the degree of cross-linking and the placement of the cross-links on the glycan strands can act in tandem to ensure that the cell wall offers sufficient resistance to propagation of cracks. Further, we suggest a possible mechanism by which peptide bond hydrolysis, via judicious cleaving of peptide cross-links, can act to mitigate this risk of failure. We also study the reinforcing effect of MreB cytoskeleton, which can offer a degree of safety to the cell wall. However, we show that the cross-linked structure of the cell wall is its primary line of defense against mechanical failure.

Chemical Synthesis of an Enzyme Containing an Artificial Catalytic Apparatus

With the goal of investigating electronic aspects of the catalysis of peptide bond hydrolysis, an analogue of HIV-1 protease was designed in which a non-peptide hydroxy-isoquinolinone artificial catalytic apparatus replaced the conserved Asp25–Thr26–Gly27 sequence in each 99-residue polypeptide chain of the homodimeric enzyme molecule. The enzyme analogue was prepared by total chemical synthesis and had detectable catalytic activity on known HIV-1 protease peptide substrates. Compared with uncatalyzed hydrolysis, the analogue enzyme increased the rate of peptide bond hydrolysis by ∼108-fold. Extensions of this unique approach to the study of enzyme catalysis in HIV-1 protease are discussed.

A Selective, Dual Emission β-Alanine Aminopeptidase Activated Fluorescent Probe for the Detection of Pseudomonas aeruginosa, Burkholderia cepacia, and Serratia marcescens.

Selective detection of β-alanyl aminopeptidase (BAP)-producing Pseudomonas aeruginosa, Serratia marcescens, and Burkholderia cepacia was achieved by employing the blue-to-yellow fluorescent transition of a BAP-specific enzyme substrate, 3-hydroxy-2-(p-dimethylaminophenyl)flavone derivative, incorporating a self-immolative linker to β-alanine. Upon cellular uptake and accumulation of the substrate by viable bacterial colonies, blue fluorescence was generated, while hydrolysis of the N-terminal peptide bond by BAP resulted in the elimination of the self-immolative linker and the restoration of the original fluorescence of the flavone derivative.

DeepCleave: a deep learning predictor for caspase and matrix metalloprotease substrates and cleavage sites.

Proteases are enzymes that cleave target substrate proteins by catalyzing the hydrolysis of peptide bonds between specific amino acids. While the functional proteolysis regulated by proteases plays a central role in the 'life and death' cellular processes, many of the corresponding substrates and their cleavage sites were not found yet. Availability of accurate predictors of the substrates and cleavage sites would facilitate understanding of proteases' functions and physiological roles. Deep learning is a promising approach for the development of accurate predictors of substrate cleavage events. We propose DeepCleave, the first deep learning-based predictor of protease-specific substrates and cleavage sites. DeepCleave uses protein substrate sequence data as input and employs convolutional neural networks with transfer learning to train accurate predictive models. High predictive performance of our models stems from the use of high-quality cleavage site features extracted from the substrate sequences through the deep learning process, and the application of transfer learning, multiple kernels and attention layer in the design of the deep network. Empirical tests against several related state-of-the-art methods demonstrate that DeepCleave outperforms these methods in predicting caspase and matrix metalloprotease substrate-cleavage sites. The DeepCleave webserver and source code are freely available at http://deepcleave.erc.monash.edu/. Supplementary data are available at Bioinformatics online.

Structural characterization of a prolyl aminodipeptidase (PepX) from Lactobacillus helveticus.

Prolyl aminodipeptidase (PepX) is an enzyme that hydrolyzes peptide bonds from the N-terminus of substrates when the penultimate amino-acid residue is a proline. Prolyl peptidases are of particular interest owing to their ability to hydrolyze food allergens that contain a high percentage of proline residues. PepX from Lactobacillus helveticus was cloned and expressed in Escherichia coli as an N-terminally His-tagged recombinant construct and was crystallized by hanging-drop vapor diffusion in a phosphate buffer using PEG 3350 as a precipitant. The structure was determined at 2.0 Å resolution by molecular replacement using the structure of PepX from Lactococcus lactis (PDB entry 1lns) as the starting model. Notable differences between the L. helveticus PepX structure and PDB entry 1lns include a cysteine instead of a phenylalanine at the substrate-binding site in the position which confers exopeptidase activity and the presence of a calcium ion coordinated by a calcium-binding motif with the consensus sequence DX(DN)XDG.

Predictive models of protease specificity based on quantitative protease-activity profiling data

Bioinformatics-based prediction of protease substrates can help to elucidate regulatory proteolytic pathways that control a broad range of biological processes such as apoptosis and blood coagulation. The majority of published predictive models are position weight matrices (PWM) reflecting specificity of proteases toward target sequence. These models are typically derived from experimental data on positions of hydrolyzed peptide bonds and show a reasonable predictive power. New emerging techniques that not only register the cleavage position but also measure catalytic efficiency of proteolysis are expected to improve the quality of predictions or at least substantially reduce the number of tested substrates required for confident predictions. The main goal of this study was to develop new prediction models based on such data and to estimate the performance of the constructed models. We used data on catalytic efficiency of proteolysis measured for eight major human matrix metalloproteinases to construct predictive models of protease specificity using a variety of regression analysis techniques. The obtained results suggest that efficiency-based (quantitative) models show a comparable performance with conventional PWM-based algorithms, while less training data are required. The derived list of candidate cleavage sites in human secreted proteins may serve as a starting point for experimental analysis.

Solution Structure of SpoIVB Reveals Mechanism of PDZ Domain-Regulated Protease Activity

Intramembrane proteases hydrolyze peptide bonds within the cell membrane as the decision-making step of various signaling pathways. Sporulation factor IV B protease (SpoIVB) and C-terminal processing proteases B (CtpB) play central roles in cellular differentiation via regulated intramembrane proteolysis (RIP) process which activates pro-σK processing at the σK checkpoint during spore formation. SpoIVB joins CtpB in belonging to the widespread family of PDZ-proteases, but much remains unclear about the molecular mechanisms and structure of SpoIVB. In this study, we expressed inactive SpoIVB (SpoIVBS378A) fused with maltose binding protein (MBP)-tag and obtained the solution structure of SpoIVBS378A from its small angle X-ray scattering (SAXS) data. The fusion protein is more soluble, stable, and yields higher expression compared to SpoIVB without the tag. MBP-tag not only facilitates modeling of the structure in the SAXS envelope but also evaluates reliability of the model. The solution structure of SpoIVBS378A fits closely with the experimental scattering data (χ2= 1.76). Comparing the conformations of PDZ-proteases indicates that SpoIVB adopts a PDZ-protease pattern similar to the high temperature requirement A proteases (HtrAs) rather than CtpB. We not only propose that SpoIVB uses a more direct and simple way to cleave the substrates than that of CtpB, but also that they work together as signal amplifiers to activate downstream proteins in the RIP pathway.

Investigation of effects of protease enzyme produced by Bacillus subtilis 168 E6-5 and commercial enzyme on physical properties of woolen fabric

Wool is one of the most important fibers in textile industry, and has been commonly used for producing value added products due to its properties of lightness, warmth, softness, and smoothness. However, the special scale structure in wool cuticle can cause felting shrinkage of wool fabrics. Proteases have been widely used to modify the surface of wool to prevent wool felting, due to their ability to catalyze the hydrolysis of peptide bonds in wool scales. Although the treatment of wool with proteases was considered as an environmentally friendly technique to provide wool fabrics with shrink resistance properties, proteases exhibited low efficacy in removing the cuticle scales because of the highly cross-linked barriers. In this study, wool fabric was treated with protease enzyme obtained from novel isolated bacteria and commercial protease enzyme, and the results were compared. The tear strength, pilling changes in ΔE values, whiteness and yellowness values of wool were controlled. Results showed that treatment with Bacillus subtilis 168 E6-5 protease enzyme yielded improvements in the physical properties of wool fabric compared with commercial enzyme.

Screening of proteolytic bacteria from tauco Surabaya based on pathogenicity and selectivity of its protease on milky fish (Chanos chanos) scales for healthy and halal collagen production

This study was reported our work on the advanced screening of proteolytic bacteria from tauco Surabaya based on pathogenicity of the isolates and the selectivity of its proteases on milky fish (Chanos chanos) scales. Determination of pathogenicity was carried out through hemolytic test on blood agar medium. Formation of clear zones around the growing bacterial colonies indicate the pathogenic bacteria, whereas no clear zones around the bacterial colonies are formed in the results of non-pathogenic bacteria test. Protease which has selectivity to milky fish scales can hydrolyse peptide bonds in milk fish scales to produce soluble collagen that can be measured as dissolved protein. The results showed that the order of pathogenicity qualitatively from the lowest to the highest were the isolate of HTcUM7.1<HTcUM6.1.1<HTcUM2<HTcUM10< HTcUM9.1<HTcUM6.2.2< HTcUM8. Crude extract of protease from HTcUM7.1, HTcUMi0 and HTcUM6.2.2 successively have specific activity of protease were 6.044±3.390, 0.311±1.099 and 1.718±0.040 U/mg. Furthermore, it was able to produce the dissolve protein with concentration as much as 0.1015±0.040, 0.0930±0.044 and 0.0825±0.004 mg/mL. Overall results showed that HTcUM7.1 was the most potential proteolytic bacteria as source of protease for production halal and healthy collagen from milky fish scale.

Engineering E. coli recombinant strains for high yield production of Burkholderia pseudomallei specific antigens

A risk of introducing into the Russian Federation exotic infections including laboratory-confirmed melioidosis regularly recorded worldwide necessitates development and improvement of express diagnostics tools. Cross reactivity between phylogenetically related species of the genus Burkholderia complicates melioidosis diagnostics by express test methods based on using monoclonal antibodies against pathogen exopolysaccharide epitopes. Searching for target antigens to create the next generation group- and species-specific immunodiagnostic reagents for identifying Burkholderia pseudomallei is still of high priority. The study was aimed at cloning complete coding sequences for cell surface proteins differentiating Burkholderia pseudomallei and optimizing recombinant antigens purification protocol. In silico comparative study allowed to select highly immunogenic B. pseudomallei outer membrane proteins Omp38 and OmpA/МotB as target biomolecules. For cloning, omp38 and ompA/motB gene-specific amplicons were obtained by PCR and ligated with the linear expression vector RIC-Ready pPAL7. Competent E. coli C-Max5 cells were transformed by a ligation mixture for producing recombinant plasmids, which were further purified to transform E. coli BL21 (DE3) cells for robust recombinant protein expression. Due to a potential multimeric protein structure, a standard protein purification protocol from native cell lysate was inefficient, which was modified to increase recombinant protein yield. However, by adding denaturing conditions at intermediate purification steps caused hydrolysis of peptide bonds in the target proteins, presumably between proline and asparagine residues. As a result, N-terminal fragments connecting recombinant proteins to the stationary phase of chromatographic column were eluted and evaluated for linear epitope detection according to their molecular weights. In silico analysis data identified highly antigenic motifs within the polypeptides studied. Thus, strains of E. coli BL21(DE3) BpsOmp39 and E. coli BL21(DE3) BpsOmpА engineered by us produce cell surface proteins Omp38 and ОmpA/МotB derived from melioidosis pathogen, which can be useful for developing diagnostic test systems.

Characterization of Legumain Degradome Confirms Narrow Cleavage Specificity

Legumain or asparagine endopeptidase is a unique cysteine endopeptidase with a distinctive specificity for the hydrolysis of peptide bonds after asparagine and to a lesser extent after aspartate. It is ubiquitously expressed in various tissues and besides its involvement in immune response and other physiological processes, it was also shown to play a role in pathological states such as inflammation and cancer. In order to improve our understanding of legumain substrate recognition we have performed proteomic profiling of legumain specificity on native proteins derived from MDA-MB-231 cells using two different N-terminal labelling methodologies (FPPS and ISIL). Our data revealed narrow cleavage specificity for P1 position combined with clear cleavage preference for unstructured secondary regions in the substrate proteins. No extended cleavage specificity on native proteins was observed. Moreover, a limited number of identified cleavages on individual substrates suggest its primary role in precision proteolysis and regulatory proteolytic events.

Optimization of protease production in indigenous Bacillus species isolated from soil samples in Lagos, Nigeria using response surface methodology

Abstract Proteases catalyse the hydrolysis of peptide bonds in proteins and offer a huge potential for application in industries, including detergent, dairy, leather, baking, pharmaceutical and beverage industries. In this study, indigenous Bacillus species were isolated from soil samples collected from abattoir, refuse and non-refuse sites in Lagos, Nigeria and optimized for protease production. The isolates were purified on Bacillus agar and screened for protease production on casein agar. Three strains showing high potential for protease production were identified as Bacillus cereus ABBA1, Bacillus subtilis RD7 and Bacillus subtilis NRD9 via amplification and analysis of 16S rRNA genes. Protease optimization was done Insilco using Box-Behnken Design (BBD) by response surface methodology (RSM) with Design-Expert software and then validated experimentally. Factors optimized include temperature, pH, carbon and nitrogen source and inoculum density. Statistical analysis was done using ANOVA. The results obtained from the Insilco experimental model revealed high protease activity of 159.43 U/ml, 141.28 U/ml and 138.17 U/ml while experimental validation generated a high protease activity of 200.56 U/ml, 176.00 U/ml and 163.76 U/ml for strains ABBA1, RD7 and NRD9, respectively in optimized medium. This corresponds to 33.54-, 42.21- and 36.64- fold increase in protease production compared to the unoptimized protease production medium. The optimum conditions for extracellular protease production obtained from quadratic model of RSM were 40 °C, pH 8.5, 2.5% (v/v) inoculum density, 1.5 g/L maltose and 2.0 g/L beef extract powder. The model prediction agreed with the experimental data (R2 = 0.98) and was statistically significant (p ≤ 0.05). This results further confirms the need to optimize the production parameters to achieve maximum yield and economical use of available resources during production of industrially important enzymes.

Proteolysis of β-lactoglobulin by Trypsin: Simulation by Two-Step Model and Experimental Verification by Intrinsic Tryptophan Fluorescence

To distinguish differences in enzymatic hydrolysis of various proteins, we propose an algorithm using a dataset of fluorescence spectra obtained at different moments of hydrolysis t. This algorithm was demonstrated in the example of β-lactoglobulin (β-LG) proteolysis by trypsin. The procedure involved processing the spectra to obtain the wavelength of the maximum fluorescence λmax, which was found to be proportional to the fraction of tryptophanes in hydrated proteolysis products (demasked tryptophanes). Then, the dependence λmax(t) was fitted by biexponential function with two exponential terms, one of which was responsible for the fast part of the fluorescence change during proteolysis. The contribution of this term was quite different for various protein substrates—it was positive for β-LG and negative for β-casein. The observed differences in proteolysis of different substrates were explained by different demasking processes. Combining the fluorescence data with the degrees of hydrolysis of peptide bonds allowed us to analyze the hydrolysis of β-LG in the framework of the two-step proteolysis model and estimate the ratio of rate constants of demasking and hydrolysis and the percentages of initially masked and resistant peptide bonds. This model predicted the existence of a bimodal demasking process with a fast part at the beginning of proteolysis and lag-type kinetics of release for some peptides. Compared with monitoring proteolysis in terms of the degree of hydrolysis only, the fluorescence data are helpful for the recognition of proteolysis patterns.

Purification and characterization of alkaline protease produced by Streptomyces flavogriseus and its application as a biocontrol agent for plant pathogens

Background A protease is an enzyme that helps proteolysis: protein catabolism by hydrolysis of peptide bonds. Proteases were the first enzymes to be commercialized, partly on the account of the history of availability and on account of need. Aim The aim of the present work is studying the properties of the partially purified enzyme and some application of the enzyme. Materials and methods In this study, alkaline protease was produced by Streptomyces flavogriseus using the shaking method. The enzyme was partially purified by ammonium sulfate precipitation. The most active fraction was evaluated on Sephadex G-100 column chromatography. The activity of the enzyme was determined at different temperatures and different pH values using various buffers. Results and conclusion In this study, alkaline protease produced by S. flavogriseus was partially purified by ammonium sulfate precipitation at 75% w/v saturation and pooled on Sephadex G-100 with a yield of 60.85% and specific activity of 85.14 U/mg protein. The enzyme was optimally active at 45°C in 0.5 mol/l potassium phosphate buffer (pH 8.0) after 30 min of incubation and was broadly stable at 45°C for 60 min and pH 6–8. The alkaline protease relative activity was increased in the presence Mn2+, Co2+, K+, Na+, Fe2+, and Mg2+ at concentration 1 mmol/l, respectively.

Analisis Kematian Larva Nyamuk Aedes agypti Akibat Pemberian Perasan Buah Nanas (Ananas comosus)

South Sumatra including 11 provinces experienced outbreaks. Aedes agypti is the main vector of dengue fever. Eradication by breaking the life cycle of Aedes agypti (larvae) mosquitoes which are not a health risk and environmentally friendly. Pineapple grows a lot in Indonesia, especially South Sumatra. Bromelin is an enzyme in pineapple which hydrolyzes peptide bonds in the protein content to amino acids. Parts of pineapple meat are used more while the skin and humps are wasted. Objective: to determine the effect of variations in concentration (100%, 75%, 50%, 25%) of pineapple juice (skin, meat, hump/stem) and the time of the death of Aedes agypti mosquito larvae. Research method: pure experimental, June-July 2018. The research subjects were 3000 Aedes agypti instar III larvae, population 2404 larvae, 2100 larvae samples, simple sampling random. Analysis of Pearson test data, One Way Anava test, post hoc LSD and post hoc Duncan, 95% confidence level. There was a relationship between the variation of pineapple juice concentration and larvae mortality with a sig (2-tailed) value of 0.001, insignificant meat and skin but skin and hump/stem or meat and hump/stem are significantly different from the number of mosquito larvae deaths in the first 12 hours of meat 97,67%, skin 96,83%, humps 87,17%. The higher the concentration of the more deaths of Aedes agypti mosquito larvae with the skin part has the same ability as meat and better than hump.

Oligopeptides Generated by Neprilysin Degradation of β-Amyloid Have the Highest Cu(II) Affinity in the Whole Aβ Family.

The catabolism of β-amyloid (Aβ) is carried out by numerous endopeptidases including neprilysin, which hydrolyzes peptide bonds preceding positions 4, 10, and 12 to yield Aβ4-9 and a minor Aβ12- x species. Alternative processing of the amyloid precursor protein by β-secretase also generates the Aβ11- x species. All these peptides contain a Xxx-Yyy-His sequence, also known as an ATCUN or NTS motif, making them strong chelators of Cu(II) ions. We synthesized the corresponding peptides, Phe-Arg-His-Asp-Ser-Gly-OH (Aβ4-9), Glu-Val-His-His-Gln-Lys-am (Aβ11-16), Val-His-His-Gln-Lys-am (Aβ12-16), and pGlu-Val-His-His-Gln-Lys-am (pAβ11-16), and investigated their Cu(II) binding properties using potentiometry, and UV-vis, circular dichroism, and electron paramagnetic resonance spectroscopies. We found that the three peptides with unmodified N-termini formed square-planar Cu(II) complexes at pH 7.4 with analogous geometries but significantly varied Kd values of 6.6 fM (Aβ4-9), 9.5 fM (Aβ12-16), and 1.8 pM (Aβ11-16). Cyclization of the N-terminal Glu11 residue to the pyroglutamate species pAβ11-16 dramatically reduced the affinity (5.8 nM). The Cu(II) affinities of Aβ4-9 and Aβ12-16 are the highest among the Cu(II) complexes of Aβ peptides. Using fluorescence spectroscopy, we demonstrated that the Cu(II) exchange between the Phe-Arg-His and Val-His-His motifs is very slow, on the order of days. These results are discussed in terms of the relevance of Aβ4-9, a major Cu(II) binding Aβ fragment generated by neprilysin, as a possible Cu(II) carrier in the brain.

Directed evolution of the 3C protease from coxsackievirus using a novel fluorescence-assisted intracellular method.

Proteases are crucial for regulating biological processes in organisms through hydrolysis of peptide bonds. Recombinant proteases have moreover become important tools in biotechnological, and biomedical research and as therapeutics. We have developed a label-free high-throughput method for quantitative assessment of proteolytic activity in Escherichia coli. The screening method is based on co-expression of a protease of interest and a reporter complex. This reporter consists of an aggregation-prone peptide fused to a fluorescent protein via a linker that contains the corresponding substrate sequence. Cleavage of the substrate rescues the fluorescent protein from aggregation, resulting in increased fluorescence that correlates to proteolytic activity, which can be monitored using flow cytometry. In one round of flow-cytometric cell sorting, we isolated an efficiently cleaved tobacco etch virus (TEV) substrate from a 1:100 000 background of non-cleavable sequences, with around 6000-fold enrichment. We then engineered the 3C protease from coxsackievirus B3 (CVB3 3Cpro) towards improved proteolytic activity on the substrate LEVLFQ↓GP. We isolated highly proteolytic active variants from a randomly mutated CVB3 3Cpro library with up to 4-fold increase in activity. The method enables simultaneous measurement of proteolytic activity and protease expression levels and can therefore be applied for protease substrate profiling, as well as directed evolution of proteases.

Stanovení aktivity proteolytických enzymů během výroby sladu

Proteolytic enzymes (proteases) are widely represented in living organisms where they catalyze the hydrolysis of peptide bonds in proteins and peptides. In malting barley, they are found in small quantities, and their activation takes place only in the process of controlled germination – malt production. In germinating barley, proteases hydrolyze storage proteins to high molecular and low molecular weight degradation products while simultaneously synthesizing other proteins. The final grain modification affects significantly quality of the produced malts and subsequently also technology and characters of the produced beer.

Single-Molecule Analyses Reveal Rhomboid Proteins Are Strict and Functional Monomers in the Membrane

Intramembrane proteases hydrolyze peptide bonds within the membrane as a regulatory paradigm that is conserved across all forms of cellular life. Many of these enzymes are thought to be oligomeric, and that their resulting quaternary interactions form the basis of their regulation. However, technical limitations have precluded directly determining the oligomeric state of intramembrane proteases in any membrane. Using single-molecule photobleaching, we determined the quaternary structure of 10 different rhomboid proteins (the largest superfamily of intramembrane proteases) and six unrelated control proteins in parallel detergent micelle, planar supported lipid bilayer, and whole-cell systems. Bacterial, parasitic, insect, and human rhomboid proteases and inactive rhomboid pseudoproteases all proved to be monomeric in all membrane conditions but dimeric in detergent micelles. These analyses establish that rhomboid proteins are, as a strict family rule, structurally and functionally monomeric by nature and that rhomboid dimers are unphysiological.

Carbon Dots/Cu2O Composite with Intrinsic High Protease‐Like Activity for Hydrolysis of Proteins under Physiological Conditions

AbstractWhereas a variety of inorganic nanomaterials possessing intrinsic peroxidase‐like activity have recently attracted considerable interest, little attention is given to explore their intrinsic protease‐mimic activities. And the construction of efficient enzyme mimetics for the hydrolysis of peptide bonds in proteins still represents a major technical challenge due to the high stability of peptide bonds and the importance of proteases in biology and industry. Herein, it is described for the first time that Cu2O‐decorated carbon quantum dots (CQDs/Cu2O) possess an intrinsic protease‐mimic activity to hydrolyze proteins including bovine serum albumin (BSA) and casein under physiological conditions. CQDs/Cu2O also features a desirable stability and good reusability. CQDs/Cu2O has a much higher affinity for BSA than trypsin. To the best of knowledge, this is the first example of protein hydrolysis by stable composite nanomaterials under physiological conditions which may open this catalytic system for a multitude of potential applications in biological systems.