A microchip-based proteolytic digestion system driven by electroosmotic pumping

Microchip-based proteomic analysis requires proteolytic digestion of proteins in microdevices. Enzyme reactors in microdevices, fabricated in glass, silicon, and PDMS substrates, have recently been demonstrated for model protein digestions. The common approach used for these enzyme reactors is employment of a syringe pump(s) to generate hydrodynamic flow, driving the proteins through the reactors. Here we present a novel approach, using electroosmotic flow (EOF) to electrokinetically pump proteins through a proteolytic system. The existence of EOF in the proteolytic system packed with immobilized trypsin gel beads was proven by imaging the movement of a neutral fluorescent marker. Digestions of proteins were subsequently carried out for 12 min, and the tryptic peptides were analyzed independently using capillary electrophoresis (CE) and MALDI-TOF mass spectrometry (MS). The results from CE analysis of the tryptic peptides from the EOF-driven proteolytic system and a conventional water bath digestion were comparable. MALDI-TOF MS was used to identify the parent protein and the tryptic peptides using MS-Fit database searching. The potential utility of the EOF-driven proteolytic system was demonstrated by direct electro-elution of proteins from an acrylamide gel into the proteolytic system, with elution and tryptic digestion achieved in a single step. The EOF-driven proteolytic system, thus, provides a simple way to integrate protein digestion into an electrophoretic micro total analysis system for protein analysis and characterization.     

Peptide mapping of polypeptides separated by two-dimensional electrophoresis: protease digestion directly on the two-dimensional gel followed by electrophoresis in reverse direction

A method is described which allows to reveal simultaneously the proteolytic patterns of numerous polypeptides separated by two-dimensional electrophoresis. After two-dimensional electrophoresis, the gels were dipped successively in buffers for preequilibration, protease digestion, and reequilibration. They were then returned to the electrophoresis tank, and electrophoresis was continued for a short time. After silver staining, digestion products appeared, lined up behind the original polypeptide spots. The method allows proteolytic patterns of numerous polypeptides to be visualized simply and quickly. Among proteins of wheat leaves, 31 groups of related polypeptides were found according to the similarity of their proteolytic patterns.     

Plant proteinases and inhibitors: an overview of biological function and pharmacological activity

Proteinases play a fundamental metabolic role during the life cycle in the plant kingdom. By interacting with endogenous or exogenous inhibitors, the proteolytic activity is modulated to meet metabolic requirements. By probing proteolytic enzymes with their inhibitors, it is possible to identify novel functions unrelated to their proteolytic activity. A group of plant proteolytic enzymes stands as a line of defence against environmental changes as their activation is triggered following various types of stress. On the other hand, plants also contain proteinase inhibitors as countermeasures for their protection against insects and pests. Both proteinases and inhibitors emerge as useful tools to combat human diseases. This review focuses on the biochemical characterization of plant proteinases, their inhibitors, the pharmacological potential of proteinases and inhibitors, and new putative emerging functions of proteolytically inhibited proteinases.     

Characterization of N-terminal processing of group VIA phospholipase A<Subscript>2</Subscript> and of potential cleavage sites of amyloid precursor protein constructs by automated identification of signature peptides in LC/MS/MS analyses of proteolytic digests

Dysregulation of proteolytic processing of the amyloid precursor protein (APP) contributes to the pathogenesis of Alzheimer's Disease, and the Group VIA phospholipase A(2) (iPLA(2)beta) is the dominant PLA(2) enzyme in the central nervous system and is subject to regulatory proteolytic processing. We have identified novel N-terminal variants of iPLA(2)beta and previously unrecognized proteolysis sites in APP constructs with a C-terminal 6-myc tag by automated identification of signature peptides in LC/MS/MS analyses of proteolytic digests. We have developed a Signature-Discovery (SD) program to characterize protein isoforms by identifying signature peptides that arise from proteolytic processing in vivo. This program analyzes MS/MS data from LC analyses of proteolytic digests of protein mixtures that can include incompletely resolved components in biological samples. This reduces requirements for purification and thereby minimizes artifactual modifications during sample processing. A new algorithm to generate the theoretical signature peptide set and to calculate similarity scores between predicted and observed mass spectra has been tested and optimized with model proteins. The program has been applied to the identification of variants of proteins of biological interest, including APP cleavage products and iPLA(2)beta, and such applications demonstrate the utility of this approach.     

PPESO3监测微波辅助酶解蛋白的进程

本实验将胰蛋白酶固定在荧光聚合物PPESO3上, 固定后的胰蛋白酶在酶解蛋白过程中通过监测荧光聚合物荧光强度的变化, 从而实现监测蛋白酶解过程.     

Time-resolved limited proteolysis of mitogen-activated protein kinase-activated protein kinase-2 determined by LC/MS only

Mass spectrometry has gained prominence in limited proteolysis studies largely due to its unparalleled precision in determining protein molecular mass. However, proteolytic fragments usually cannot be identified through direct mass measurement, since multiple subsequences of a protein can frequently be matched to observed masses of proteolytic fragments. Therefore, additional information from N-terminal sequencing is often needed. Here we demonstrate that mass spectrometry analysis of the time course of limited proteolysis reactions provides new information that is self-sufficient to identify all proteolytic fragments. The method uses a non-specific protease like subtilisin and exploits information contained in the time-resolved dataset such as: increased likelihood of identifying larger fragments generated during initial proteolysis solely by their masses, additivity of the masses of two mutually exclusive sequence regions that generate the full-length molecule (or an already assigned subfragment), and analyses of the proteolytic subfragment patterns that are facilitated by having established the initial cleavage sites. We show that the identities of the observed proteolytic fragments can be determined by LC/MS alone because enough constraints exist in the time-resolved dataset. For a medium-sized protein, it takes about 8 h to complete the study, a significant improvement over the traditional SDS-PAGE and N-terminal sequencing method, which usually takes several days. We illustrate this method with application to the catalytic domain of mitogen-activated protein kinase-activated protein kinase-2, and compare the results with N-terminal sequencing data and the known X-ray crystal structure.     

Combination of zymography and immunodetection to analyze proteins in complex culture supernatants

The physiological function of an allergen might be an important factor for the allergenicity. The major grass pollen allergen Phl p 1 shows sequence similarities to the consensus sequences of cysteine proteases. However, up to now, the proteolytic activity of Phl p 1 is controversial. The culture supernatant of Phl p 1-transfected clones from Pichia pastoris showed a proteolytic activity but this might be due to Phl p 1 or irrelevant yeast contaminants. To solve this question, we made use of the zymogram technique and improved it. Substrate as well as substrate concentration was changed from 1% casein to 0.25% skimmed milk powder. For staining, we used a colloidal Coomassie stain (RotiBlue) with a higher sensitivity and better practicability than the conventional Coomassie staining. The proteins in the zymogels and in the SDS-PAGE gels showed similar electrophoretic mobility. Furthermore, the zymogels could be blotted and immunostained. Thus, the molecular mass of the proteolytic bands could be determined and directly compared with immunoblotting results. To clearly assign the protease, we separated the culture supernatant of the Phl p 1-transfected P. pastoris clone by affinity chromatography with monoclonal antibody. Our studies demonstrate that the proteolytic activity did not belong to the recombinant allergen but to the yeast proteins. The enzyme was classified by zymogram inhibition tests as a strong serine protease.     

Characteristics of the Proteolytic Enzymes Produced by Lactic Acid Bacteria

Over the past several decades, we have observed a very rapid development in the biotechnological use of lactic acid bacteria (LAB) in various branches of the food industry. All such areas of activity of these bacteria are very important and promise enormous economic and industrial successes. LAB are a numerous group of microorganisms that have the ability to ferment sugars into lactic acid and to produce proteolytic enzymes. LAB proteolytic enzymes play an important role in supplying cells with the nitrogen compounds necessary for their growth. Their nutritional requirements in this regard are very high. Lactic acid bacteria require many free amino acids to grow. The available amount of such compounds in the natural environment is usually small, hence the main function of these enzymes is the hydrolysis of proteins to components absorbed by bacterial cells. Enzymes are synthesized inside bacterial cells and are mostly secreted outside the cell. This type of proteinase remains linked to the cell wall structure by covalent bonds. Thanks to advances in enzymology, it is possible to obtain and design new enzymes and their preparations that can be widely used in various biotechnological processes. This article characterizes the proteolytic activity, describes LAB nitrogen metabolism and details the characteristics of the peptide transport system. Potential applications of proteolytic enzymes in many industries are also presented, including the food industry.     

Extracellular proteolytic processing ofAspergillus awamori GAI into GAII is supported by physico-chemical evidence

The proportion of glucoamylases, GAI and GAII, in the culture supernatant ofAspergillus awamori fermentations depends on the medium C/N ratio in such a way that the transformation of GAI into GAII is favored by the existence of a surplus of the carbon source in the growth medium. This condition also favors the appearance of the proteolytic activity. The authors report the observation that the shift in the isoenzyme proportion was concomitant to the peak of proteolytic activity. A peptide that may have resulted from the continuous degradation of the GAI C-terminal peptide, Gp-1, was isolated by gel filtration and purified by reversephase chromatography. This peptide matched with the region G¹⁴-A³⁴ of the substrate-binding domain of GAI, thus reinforcing the hypothesis of the extracellular proteolytic processing of GAI.     

Variations in some extracellular enzyme activities during degradation of lignocellulose byPhanerochaete chrysosporium

The white-rot fungusPhanerochaete chrysosporium is able to degrade lignin only when its primary growth phase is completed. We have recently shown that the organism is able to establish new growth at 10–15 d intervals by recycling its own nitrogen (2). We have now further characterized this growth-rest cycle by measuring changes in extracellular protease, cellulase, and xylanase activities together with total extracellular protein during growth on different carbon sources.

1. WhenP. chrysosporium is grown on a N-limited glucose medium, the cessation of primary growth is closely connected to the increase in extracellular proteolytic activity. When the culture is not O₂-limited (2) it becomes ligninolytically active after about 2 d with a simultaneous decrease in proteolytic activity and an increase in extracellular protein. In O₂-limited cultures, the proteolytic activity remains on a high level for up to 6–7 d. During the second growth phase, the proteolytic activity again increases.
2. When P.chrysosporium is grown on a N-limited glucose medium supplemented with lignocellulosic materials the cellulase and xylanase activities are suppressed and the growth is again connected to an increase in extracellular proteolytic activity. Lignin is not degraded during the growth phases.
3. When P.chrysosporium is grown on a N-limited medium with lignocellulose as the only energy source, the growth phases are connected with increased cellulolytic, xylanolytic, and proteolytic activities. Again during the growth phase, lignin is not degraded. During the ligninolytic phase the level of measured extracellular enzyme activities decreases. A simultaneous increase in total extracellular protein seems to indicate that these enzymes are partly reused for synthesis of the ligninolytic system. Proteins associated with the ligninolytic system appear to be partly reused to synthesize the hydrolytic enzymes for the next growth phase.

     

Partial purification and characterization of two proteases fromAgave fourcroydes

Large amounts of wastes are produced by the henequen industry from which it may be possible to obtain products of commercial importance. Our laboratory has detected proteolytic activity inAgave fourcroydes (henequen) juice. The proteolytic activity is a result of two proteases of 14,500 and 12,000 daltons, respectively. Both enzymes are very stable at -20°C., when freeze-dried and in the presence of EDTA and cysteine. They differ in pH optimum and thermal stability.     

Enzyme‐Degradable Self‐Assembled Hydrogels From Polyalanine‐Modified Poly(ethylene glycol) Star Polymers

The generation of a range of star‐shaped block copolymers composed of a biocompatible poly(ethylene glycol) (PEG) core tethered to a polyalanine (PAla) shell that possesses the capability to (reversibly) self‐assemble in water is described. The hydrogels formed offer a hydrophilic environment ideal for biological processes involving proteins and are able to withhold albumin for prolonged periods before its triggered release following the targeted material degradation by the proteolytic enzyme elastase. Consequently, the materials formed offer significant promise for the delivery of proteins, and possibly inhibitors, in response to a proteolytic enzyme overexpressed in chronic wounds.     

Effect of bioengineering lacticin 3147 lanthionine bridges on specific activity and resistance to heat and proteases

Lacticin 3147 is a lantibiotic with seven lanthionine bridges across its two component peptides, Ltnα and Ltnβ. Although it has been proposed that the eponymous lanthionine and (β-methyl)lanthionine (Lan and meLan) bridges present in lantibiotics make an important contribution to protecting the peptides from thermal or proteolytic degradation, few studies have investigated this link. We have generated a bank of bioengineered derivatives of lacticin 3147, in which selected bridges were removed or converted between Lan and meLan, which were exposed to high temperature or proteolytic enzymes. Although switching Lan and meLan bridges has variable consequences, it was consistently observed that an intact N-terminal lanthionine bridge (Ring A) confers Ltnα with enhanced resistance to thermal and proteolytic degradation.     

Protease Probes that Enable Excimer Signaling upon Scission

Peptide‐based probes that fluoresce upon proteolytic cleavage are invaluable tools for monitoring protease activity. The read‐out of protease activity through pyrene excimer signaling would be a valuable asset because the large Stokes shift and the long lifetime of the excimer emission facilitate measurements in autofluorescent media such as blood serum. However, proteolytic cleavage abolishes rather than installs the proximity relationships required for excimer signaling. Herein, we introduce a new probe architecture to enable the switching on of pyrene excimer emission upon proteolytic scission. The method relies on hairpin‐structured peptide nucleic acid (PNA)/peptide hybrids with pyrene units and anthraquinone‐based quencher residues positioned in a zipper‐like arrangement within the PNA stem. The excimer hairpin peptide beacons afforded up to a 50‐fold enhancement of the pyrene excimer emission. Time‐resolved measurements allowed the detection of matrix metalloprotease 7 in human blood serum.     

Down syndrome critical region 1 enhances the proteolytic cleavage of calcineurin

Down syndrome critical region 1 (DSCR1), an oxidative stress-response gene, interacts with calcineurin and represses its phosphatase activity. Recently it was shown that hydrogen peroxide inactivates calcineurin by proteolytic cleavage. Based on these facts, we investigated whether oxidative stress affects DSCR1-mediated inactivation of calcineurin. We determined that overexpression of DSCR1 leads to increased proteolytic cleavage of calcineurin. Convertsely, knockdown of DSCR1 abolished calcineurin cleavage upon treatment with hydrogen peroxide. The PXIIXT motif in the COOH-terminus of DSCR1 is responsible for both binding and cleavage of calcineurin. The knockdown of overexpressed DSCR1 in DS fibroblast cells also abrogated calcineurin proteolysis by hydrogen peroxide. These results suggest that DSCR1 has the ability to inactivate calcineurin by inducing proteolytic cleavage of calcineurin upon oxidative stress.     

Proteolytic Activities of Kiwifruit Actinidin (Actinidia deliciosa cv. Hayward) on Different Fibrous and Globular Proteins: A Comparative Study of Actinidin with Papain

Actinidin, a member of the papain-like family of cysteine proteases, is abundant in kiwifruit. To date, a few studies have been provided to investigate the proteolytic activity and substrate specificity of actinidin on native proteins. Herein, the proteolytic activity of actinidin was compared to papain on several different fibrous and globular proteins under neutral, acidic and basic conditions. The digested samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and densitometry to assess the proteolytic effect. Furthermore, the levels of free amino nitrogen (FAN) of the treated samples were determined using the ninhydrin colorimetric method. The findings showed that actinidin has no or limited proteolytic effect on globular proteins such as immunoglobulins including sheep IgG, rabbit IgG, chicken IgY and fish IgM, bovine serum albumin (BSA), lipid transfer protein (LTP), and whey proteins (α-lactalbumin and β-lactoglobulin) compared to papain. In contrast to globular proteins, actinidin could hydrolyze collagen and fibrinogen perfectly at neutral and mild basic pHs. Moreover, this enzyme could digest pure α-casein and major subunits of micellar casein especially in acidic pHs. Taken together, the data indicated that actinidin has narrow substrate specificity with the highest enzymatic activity for the collagen and fibrinogen substrates. The results describe the actinidin as a mild plant protease useful for many special applications such as cell isolation from different tissues and some food industries as a mixture formula with other relevant proteases.     

X-ray absorption near edge structure study on Acutolysin-C,a zinc-metalloproteinase from Agkistrodon acutus venom: Insight into the acid-inactive mechanism

Acutolysin-C, a snake-venom zinc metalloproteinase, displays a distinct pH-dependent proteolytic activity, which has been tentatively assigned to a structural change of the zinc-containing catalytic center. In this work we compare X-ray absorption near-edge structure (XANES) experimental spectra at the Zn K-edge and theoretical calculations of solutions at different pH values. The experimental data show clear differences confirmed by a best fit using the MXAN procedure. The results show that, when pH decreases from pH 8.0 to pH 3.0, the zinc-coordinating catalytic water molecule moves far from the Glu143 residue that is considered to play an essential role in the proteolytic process. Data suggests that this is the possible mechanism that deactivates the metalloproteinase.     

Rapid,sensitive fluorimetric assay for proteases using fluorescein-labelled albumin coupled to Fractogel activated with 2-fluoro-1-methylpyridinium salt in flow-injection analysis

A rapid and sensitive flow-injection method is described for the fluorimetric determination of the proteolytic activity of a number of enzymes. The substrate, fluorescein-labelled bovine serum albumin coupled to a 2-fluoro 1-methylpyridinium salt-activated Fractogel support, is packed in a small reactor (35 × 2 mm i.d.) and inserted into a flow manifold. Under the reaction conditions the amount of fluorescein released from enzymatic cleavage of the substrate is found to be directly proportional to the activity of the proteolytic enzyme. Sample throughput is 30–35 h^?1. The calibration range for trypsin is linear up to at least 0.35 μg ml^?1. Linear responses were also obtained for otherproteolytic enzymes such as papain, chymotrypsin, ficin and bromelain.     

Enhancing the Cell Permeability and Metabolic Stability of Peptidyl Drugs by Reversible Bicyclization

Therapeutic applications of peptides are currently limited by their proteolytic instability and impermeability to the cell membrane. A general, reversible bicyclization strategy is now reported to increase both the proteolytic stability and cell permeability of peptidyl drugs. A peptide drug is fused with a short cell‐penetrating motif and converted into a conformationally constrained bicyclic structure through the formation of a pair of disulfide bonds. The resulting bicyclic peptide has greatly enhanced proteolytic stability as well as cell‐permeability. Once inside the cell, the disulfide bonds are reduced to produce a linear, biologically active peptide. This strategy was applied to generate a cell‐permeable bicyclic peptidyl inhibitor against the NEMO‐IKK interaction.     

Conformation, activity and proteolytic stability of acid phosphatase on clay minerals and soil colloids from an Alfisol

The present study was carried out to investigate the conformation, enzymatic activity and proteolytic stability of acid phosphatase on montmorillonite, kaolinite and soil colloids from an Alfisol by means of circular dichroism (CD) spectroscopy, isothermal titration microcalorimetry (ITC) and biochemical assay, respectively. The results showed that the secondary structure of phosphatase was changed from disordered type to ordered form during adsorption/desorption cycle, organic substance and 2:1-clay mineral in Brown Soil benefited the formation of ordered structure. Enzymatic activity of phosphatase was inhibited while the proteolytic stability was promoted after the interaction with active particles from permanent charge soil. The decrease of enzymatic activity and the increase of proteolytic stability resulted by montmorillonite and organic colloid were both greater than that by kaolinite and inorganic colloid, which was in consistent with the extent of structural change induced by different colloid particles. Thus, one of the most significant factors responsible for the variation of enzymatic activity and proteolytic stability might be the hiding or even damage of active sites and the irrecognition of cleavage sites in enzyme molecules induced by the formation of ordered structure. The information obtained in this study is of crucial significance for the understanding of the behavior and fate of extracellular enzymes in soils with permanent charges.