Increase In Endopeptidase Activity Research Articles

Upregulation of gut cathepsin L during Eimeria tenella infection

Coccidiosis is a disease caused by Eimeria, which represents the first parasitic disease in poultry farming. Among them, E. tenella is a virulent species which specifically colonizes the caecum. The inflammatory response to infection is associated to numerous host proteases including cysteine cathepsins that can be deleterious for tissue and innate immunity integrity. Here, germ-free and conventional chickens were used as models to find out whether the microbiota could modify the intestinal expression of host cysteine cathepsins during coccidiosis. The basal caecal peptidase activity primarily relies on host proteases rather than proteases from the commensal flora. While mRNA levels of E. tenella cathepsins B and L remained unchanged in germ-free and conventional broilers, an overall increase in endopeptidase activity of cysteine cathepsins was found in E. tenella-infected caeca in both experimental models (P < 0.005). A significant decrease in avian cystatin C transcription was also observed in infected conventional, but not in infected germ-free broilers. Despite an unchanged mRNA level of avian cathepsin L (CatL), its protein expression raised following infection, in parallel with an increased transcription of antimicrobial β-defensins (AvBD1, AvBD2, AvBD4, AvBD6, and AvBD7). Taken together, data support that host CatL is post-translationally upregulated during E. tenella infection, and thus may be involved in the alteration of the gut proteolytic balance. Furthermore, CatL may participate to inflammation occurring during coccidiosis through its known ability to proteolytically inactivates up-regulated avian β-defensins that are key molecules of innate immunity.

Autodigestion and Peptidase Activity in Low Quality Baltic Sprat (Sprattus sprattus balticus Schneider, 1908)

ABSTRACTThis study was conducted on fresh and frozen-thawed sprat of the lowest utility, which is not used for consumption. The peptidase activity was measured (i) against acid- and base-hemoglobin (GPA) and as (ii) autodigestion with an effect of NaCl concentration in the range of 5–30% and a pH effect in the range of 3.5–8.0. The activity has been shown to be divided into endo and exopeptidases. The optimum endopeptidase activity in whole fresh fish was observed at pH 8.0, whereas for exopeptidases it was observed at 5.0. Under acidic conditions, the activity of endopeptidases from intestines was the same as that from carcasses, but under basic conditions, it was over 130 times higher. After freezing the sprat, the GPA was higher than in fresh spat. The increase in GPA was significant especially for endopeptidases. Freezing also affected the activity of endo and exopeptidases. The addition of 5% salt to the whole fresh sprat caused an increase in endopeptidase activity, but higher NaCl concentrations decreased it. At 20% concentration, the endopeptidase activity was almost 20 times lower than that at 5% salt. The salt concentration did not significantly affect the sprat exopeptidase activity. Sprat with the lowest utility has high peptidase activity and can produce hydrolysates used in food technology.

Chilling Stress Accelerates Degradation of Seed Storage Protein and Photosynthetic Protein during Cotton Seed Germination

AbstractLow temperature is one of the major abiotic stresses limiting the productivity and geographical distribution of many important crops. To gain a better understanding of chilling stress responses in cotton (Gossypium hirsutum L.), we carried out a comparative proteomic analysis. Cotyledon proteins of 1‐week‐old cotton seedlings treated with or without chilling treatment at 4 °C were extracted, separated by a two‐dimensional gel electrophoresis and compared. Among 1500 protein spots reproducibly detected on each gel, 25 protein spots were down‐regulated and 29 were up‐regulated. Seven cotton proteins were identified by mass spectrometry analysis as beta‐globulin A precursor fragments. One was identified as a Rubisco large subunit fragment, providing evidence of both seed storage protein and photosynthetic protein destruction by chilling stress. The related physiological and biochemical analysis showed that there was a significant increase in endopeptidase activity and activated oxygen generation rate, and an obvious decrease in carboxylation efficiency and maximum net photosynthetic rate of cotton seedlings under chilling stress. Based on the above results, the degradation mechanisms of beta‐globulin and Rubisco under chilling stress were discussed. In conclusion, our study provides new insights into chilling‐stress responses in cotton and demonstrates the advantages of proteomic analysis.

Major increase in endopeptidase activity of human cathepsin B upon removal of occluding loop contacts.

The main feature distinguishing cathepsin B from other cysteine proteases of the papain family is the presence of a large insertion loop, termed the occluding loop, which occupies the S' subsites of the enzyme. The loop is held in place mainly by two contacts with the rest of the enzyme, involving residues His110 and Arg116 on the loop that form salt bridges with Asp22 and Asp224, respectively. The influence of this loop on the endopeptidase activity of cathepsin B has been investigated using site-directed mutagenesis and internally quenched fluorogenic (IQF) substrates. Wild-type cathepsin B displays poor activity against the substrates Abz-AFRSAAQ-EDDnp and Abz-QVVAGA-EDDnp as compared to cathepsin L and papain. Appreciable increases in kcat/KM were observed for cathepsin B containing the single mutations D22A, H110A, R116A, and D224A. The highest activity however is observed for mutants where both loop to enzyme contacts are disrupted. For the triple-mutant D22A/H110A/R116A, an optimum kcat/KM value of 12 x 10(5) M-1 s-1 was obtained for hydrolysis of Abz-AFRSAAQ-EDDnp, which corresponds to a 600-fold increase relative to wild-type cathepsin B and approaches the level of activity observed with cathepsin L or papain. By comparison, the mutations have little effect on the hydrolysis of Cbz-FR-MCA. The influence of the mutations on the pH dependency of activity also indicates that the complexity of pH activity profiles normally observed for cathepsin B is related to the presence of the occluding loop. The major increase in endopeptidase activity is attributed to an increase in loop "flexibility" and suggests that the occluding loop might move when an endopeptidase substrate binds to the enzyme. The possible contribution of these interactions in regulating endopeptidase activity and the implications for cathepsin B activity in physiological or pathological conditions are discussed.

Low fluences of ultraviolet irradiation stimulate HeLa cell surface aminopeptidase and candidate "TGF alpha ase" activity.

Several forms of perturbation result in the release of bioactive molecules into the microenvironment of injured cells to mediate the inflammatory or reparative reactions which restore normal tissue structure and function. Amongst other products, ultraviolet irradiation (UV) causes the release of the growth factor TGF alpha from a variety of epithelial cell sources, apparently by a post-translational mechanism. Here we have explored the hypothesis that UV results in the activation of cell surface proteases which may then be capable of excising mature TGF alpha from its plasma membrane-bound precursor. Using a recently described, sensitive assay of peptidase activity tailored to the substrate requirements for cleavage of the scissile bonds in proTGF alpha, we have found that nonlethal fluences of UV (< 12 Jm-2) to HeLa cell cultures are followed by large increases in cell surface proteolytic activities. Amongst these, endopeptidase activity produces a similar product profile from the nonapeptide substrate to that of human leukocyte elastase, an enzyme previously shown to be capable of releasing a bioactive, mature form of TGF alpha from its cell-bound precursor. However, in addition to this candidate "TGFase" activity, cell surface aminopeptidase activity was also very significantly increased. The increase in the two classes of peptidase function differed in the timing of their responses. Aminopeptidase activation occurred immediately following UV, peaking after some 15-20 h, whereas the increase in endopeptidase activity lagged 6 h behind, cresting after 20-24 h. No evidence for a role for aminopeptidase in the activation of the endopeptidase could be found. Also, there was no increase in the total proteolytic activity demonstratable in cell extracts following UV. Attempts to interrupt the UV peptidase activation by inhibiting protein synthesis with cycloheximide were unsuccessful; rather, the inhibitor itself caused an increase in both classes of peptidase activity during the first 20 h. Unlike the UV response, both the aminopeptidase and endopeptidase ectoactivities increased simultaneously within a few hours of introducing cycloheximide into the medium of unirradiated cultures. The cycloheximide induced activity peaked after 20 h. Interestingly, cycloheximide alone has previously been shown to potentiate TGF alpha release from a cell line producing its precursor constitutively. These data suggest that both UV and cycloheximide can initiate reactions in HeLa cells which result in ectopeptidase activation of a global nature. Since both agents result in rapid interruption of DNA synthesis, it is possible that this cell surface proteolytic response may be analogous to, or part of, the "mammalian genetic stress response".(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of changed source/sink relations on proteolytic activities and on nitrogen mobilization in field-grown wheat (Triticum aestivum L.)

Nitrogen mobilization and the pattern of proteolytic enzymes were investigated in leaves and glumes of field-grown winter wheat (Triticum aestivum L.) during maturation. Source/sink relations were changed by removal of the ear, the flag leaf or the lower leaves shortly after anthesis. Removal of the ear was most effective, resulting in delayed senescence of the flag leaf with the chlorophyll, aminopeptidase and carboxypeptidase activities remaining high in contrast to the control, whereas neutral endopeptidase activity increased more slowly. No major changes were observed in the second leaf from the top in plants with either ears or flag leaves removed. Nitrogen mobilization and proteolytic activities in glumes and the remaining leaves were influenced only slightly by leaf removal. In earless plants, nitrogen was transported from the second leaf into the leaf sheath and stem, but in the flag leaf the total reduced nitrogen remained high and free amino groups increased. The increase in endopeptidase activity was influenced by the source/sink relations. However, the accumulation of amino groups and the increasing endopeptidase activity in the flag leaf of earless plants suggest that the nitrogen sink capacity did not greatly control protein degradation; it remains to be seen whether phytohormones, accumulated amino acids or other factors delayed the increase in endopeptidase activity.

Partial Characterization of a Protease Inhibitor Which Inhibits the Major Endopeptidase Present in the Cotyledons of Mung Beans

Germination of mung beans (Phaseolus aureus, Roxb.) is accompanied by an increase in the activity of the endopeptidase involved in storage protein metabolism. Enzyme activity in the cotyledons increases 25-fold during the first 5 days of germination. The cotyledons also contain inhibitory activity against the endopeptidase, and this inhibitory activity declines during germination, suggesting that inhibitors may play a role in regulating the activity of the endopeptidase.The inhibitory activity against the mung bean endopeptidase is due to the presence of two inhibitors which can be separated by chromatography on Sephadex G-100. The two inhibitors have approximate molecular weights of 12,000 and smaller than 2,000 daltons. The large inhibitor coelutes with trypsin inhibitor on Sephadex G-100, but these two inhibitory activities can be separated by means of a trypsin affinity column.The inhibitory activity disappears slowly from crude extracts incubated at 6 C and more rapidly when the extracts are incubated at 25 C or 37 C. The disappearance of inhibitory activity is accompanied by a rise of the endopeptidase activity, but an examination of the kinetics of these two phenomena suggests that they are not causally related. Fractionation of the cellular organelles on sucrose gradients shows that the inhibitory activity is not associated with the protein bodies, but rather with the cytosol. Our results suggest that the endopeptidase inhibitor(s) does not regulate the increase in endopeptidase activity which accompanies germination or the metabolism of storage protein. We, therefore, postulate that the inhibitor(s) may function in protecting the cytoplasm from accidental rupturing of the protease-containing protein bodies.

Control of Storage Protein Metabolism in the Cotyledons of Germinating Mung Beans: Role of Endopeptidase

The autodigestive proteolytic activity of extracts of cotyledons of mung beans (Phaseolus aureus Roxb.) increased 4- to 5-fold during germination. A similar increase was found in the ability of these extracts to digest added casein or mung bean globulins. The increase occurred after a 2-day lag during the next 2 to 3 days of germination and coincided with the period of rapid storage protein breakdown. To understand which enzyme(s) may be responsible for this increase in proteolytic activity, the hydrolytic activity of cotyledon extracts toward a number of synthetic substrates and proteins was measured. Germination was accompanied by a marked decline in leucine aminopeptidase, while carboxypeptidase increased about 50%. There were no dramatic changes in either alpha-mannosidase or N-acetyl-beta-glucosaminidase, enzymes which may be involved in the metabolism of the carbohydrate moieties of the reserve glycoproteins. The increase in general proteolytic activity was closely paralleled by a 10-fold increase in endopeptidase activity. This activity was inhibited by sulfhydryl reagents such as N-ethylmaleimide. Studies with inhibitors of proteolytic enzymes showed that reagents which blocked sulfhydryl groups also inhibited the rise in general proteolytic activity. Our results suggest that the appearance of a sulfhydryl-type endopeptidase activity is a necessary prerequisite for the rapid metabolism of the reserve proteins which accompanies germination.

CHANGES IN THE CONTENTS OP BARLEY PROTEOLYTIC INHIBITORS DURING MALTING AND MASHING

Barley contains three types of large-molecular proteolytic inhibitors, viz., inhibitors active against malt endopeptidases, a specific trypsin inhibitor, and a group of inhibitors of microbial proteinases and chymotrypsin. The inhibitors of malt endopeptidases disappear at an early stage of germination, just before the endopeptidase activity begins to increase. The total amount of these inhibitors is so small, however, that the great increase in endopeptidase activity occurring during germination cannot be due to the breakdown of an “equivalent” amount of inhibitors. The contents of the other two types of inhibitor in barley are much higher; they decrease slowly during germination, but high activities are still present in malt. The inhibitors of microbial proteinases are progressively inactivated during mashing. The trypsin inhibitor retains full activity through mashing, but is completely inactivated during boiling and hopping.