Trypsin-like Activity Research Articles

Antioxidant activity of the peroxidase system in cell nuclei during germination of wheat seedlings

The dynamics of the antioxidant activity of the peroxidase system in the structures of cell nuclei during the induction of germination of wheat seedlings was tracked, which was synchronized with nuclear trypsin-like activity and mitotic activity in cells. The dynamics of the antioxidant activity of the peroxidase system in the fractions of cell nuclei seems to be related to the processes of cell division and stretching during the development of plant seedlings. Previously, these results can be considered as one of the complex multilevel regulatory networks of proteinases formed in cell nuclei, which can probably participate in the turnover of oxidized proteins.

Gallic Acid Attenuates Angiotensin II-Induced Hypertension and Vascular Dysfunction by Inhibiting the Degradation of Endothelial Nitric Oxide Synthase.

Hypertension is a major cause of heart attack and stroke. Our recent study revealed that gallic acid (GA) exerts protective effects on pressure overload-induced cardiac hypertrophy and dysfunction. However, the role of GA in angiotensin II (Ang II)-induced hypertension and vascular remodeling remains unknown. C57BL/6J mice were subjected to saline and Ang II infusion. Systolic blood pressure was measured using a tail-cuff system. Vascular remodeling and oxidative stress were examined by histopathological staining. Vasodilatory function was evaluated in the aortic ring. Our findings revealed that GA administration significantly ameliorated Ang II-induced hypertension, vascular inflammation, and fibrosis. GA also abolished vascular endothelial dysfunction and oxidative stress in Ang II-infused aortas. Mechanistically, GA treatment attenuated Ang II-induced upregulation of the immunoproteasome catalytic subunits β2i and β5i leading to reduction of the trypsin-like and chymotrypsin-like activity of the proteasome, which suppressed degradation of endothelial nitric oxide synthase (eNOS) and reduction of nitric oxide (NO) levels. Furthermore, blocking eNOS activity by using a specific inhibitor (L-N G-nitroarginine methyl ester) markedly abolished the GA-mediated beneficial effect. This study identifies GA as a novel immunoproteasome inhibitor that may be a potential therapeutic agent for hypertension and vascular dysfunction.

Identification of six novel alternative transcripts of the human kallikrein-related peptidase 15 (KLK15), using 3'RACE and high-throughput sequencing.

The kallikrein-related peptidase 15 (KLK15) gene is a member of the largest cluster of serine proteases in the human genome. Exhibiting trypsin-like activity, KLK15 is most likely involved in the activation of prostate-specific antigen (PSA; also known as KLK3), an established biomarker for the diagnosis and screening of prostate cancer. High mRNA expression levels of KLK15 have already been reported in ovarian and prostate cancer, in contrast with breast cancer, where KLK15 has been proposed as a biomarker of favorable prognosis. In this study, we exploited the next-generation sequencing (NGS) technology along with 3' rapid amplification of cDNA ends (3' RACE) to discover alternative KLK15 splice variants. Extensive computational analysis of the obtained NGS data revealed the existence of novel splice junctions, thus supporting the existence of novel KLK15 transcripts. Six novel KLK15 splice variants were identified and verified by Sanger sequencing. Two of them (KLK15 v.11 and v.12) contain an open reading frame and are hence predicted to encode two novel KLK15 protein isoforms. Expression analysis of each KLK15 splice variant in sixteen cDNA pools from malignant cell lines and in normal cell lines (HEK293, HaCaT, and BJ cells) revealed very different expression profiles of particular KLK15 transcripts. Moreover, the new KLK15 splice variants were shown to be expressed in breast, ovarian, prostate, urinary bladder, colon, and renal tissue specimens. Due to the prominent clinical value of KLK15 mRNA expression, the novel KLK15 transcripts appear as candidate cancer biomarkers for diagnostic and/or prognostic purposes and, therefore, merit further investigation.

Evaluation of the insecticidal activity of Moringa oleifera seed extract and lectin (WSMoL) against Sitophilus zeamais

The maize weevil Sitophilus zeamais is a pest of remarkable economic importance as it attacks stored grains, processed cereals, and industrial foods. In this study, a water extract (WE) from Moringa oleifera seeds and water-soluble lectin (WSMoL) isolated from it were investigated for insecticidal activity against S. zeamais adults. The insects were maintained for 10 days on plates containing an artificial diet composed of wheat flour supplemented with WE (60.0–640.0 mg/g) or WSMoL (0.5–60 mg/g). Mortality rate, insect biomass, and amount of food consumed were recorded. The effects of WE and WSMoL on trypsin-like, α-amylase, and endoglucanase activities from insect guts were also investigated. Phytochemical analysis revealed the presence of flavonoids, tannins, saponins, phenylpropanoids, alkaloids, and reducing sugars in the WE. Furthermore, the presence of rutin and ellagic acid was confirmed by HPLC. The extract was toxic to S. zeamais (LC50: 214.6 mg/g) while WSMoL caused slight mortality (12.0 ± 2.7%) at the highest tested dose (60 mg/g). The intake of WE exerted moderate to strong deterrent effects. Moreover, WSMoL decreased the relative biomass gain rate and efficiency in the conversion of ingested food but did not exert deterrent action. The WE stimulated α-amylase and endoglucanase activities, while WSMoL stimulated trypsin-like activity from S. zeamais gut. In conclusion, the WE was toxic to S. zeamais probably due to its feeding-deterrent activity, while WSMoL did not cause acute mortality but negatively affected the nutritional physiology of the insects, which can have long-term impacts.

The Chymase Mouse Mast Cell Protease-4 Regulates Intestinal Cytokine Expression in Mature Adult Mice Infected with Giardiaintestinalis.

Mast cells have been shown to affect the control of infections with the protozoan parasite Giardia intestinalis. Recently, we demonstrated that Giardia excretory-secretory proteins inhibited the activity of the connective tissue mast cell-specific protease chymase. To study the potential role of the chymase mouse mast cell protease (mMCP)-4 during infections with Giardia, mMCP-4+/+ and mMCP-4−/− littermate mice were gavage-infected with G. intestinalis trophozoites of the human assemblage B isolate GS. No significant changes in weight gain was observed in infected young (≈10 weeks old) mMCP-4−/− and mMCP-4+/+ littermate mice. In contrast, infections of mature adult mice (>18 weeks old) caused significant weight loss as compared to uninfected control mice. We detected a more rapid weight loss in mMCP-4−/− mice as compared to littermate mMCP-4+/+ mice. Submucosal mast cell and granulocyte counts in jejunum increased in the infected adult mMCP-4−/− and mMCP-4+/+ mice. This increase was correlated with an augmented intestinal trypsin-like and chymotrypsin-like activity, but the myeloperoxidase activity was constant. Infected mice showed a significantly lower intestinal neutrophil elastase (NE) activity, and in vitro, soluble Giardia proteins inhibited human recombinant NE. Serum levels of IL-6 were significantly increased eight and 13 days post infection (dpi), while intestinal IL-6 levels showed a trend to significant increase 8 dpi. Strikingly, the lack of mMCP-4 resulted in significantly less intestinal transcriptional upregulation of IL-6, TNF-α, IL-25, CXCL2, IL-2, IL-4, IL-5, and IL-10 in the Giardia-infected mature adult mice, suggesting that chymase may play a regulatory role in intestinal cytokine responses.

Sputum trypsin-like protease activity relates to clinical outcome in cystic fibrosis

BackgroundIn cystic fibrosis (CF) airways excessive levels of serine trypsin-like proteases (TLPs) activate the epithelial sodium channel (ENaC) resulting in airways dehydration and promotion of mucus secretion. Despite this the relationship of TLP activity and clinical outcome has not been studied. MethodsWe analysed supernatant (sol) prepared from CF sputum from adult CF patients in two study cohorts (29 and 33 samples, respectively). Protease activities were determined by measuring the hydrolysis of peptide-based substrates or by ELISA. Lung function was assessed by spirometry (FEV1). Mortality data was retrospectively obtained and time in months until death or transplantation used for subsequent survival analysis. ResultsTLP activity inversely correlated with percent predicted FEV1 (r = −0.4, p = 0.03) and was greater in individuals who did not survive beyond 5-years from the time of sample collection. A Kaplan-Meier analysis demonstrated significantly reduced survival (p = 0.04) for individuals with high TLP activity [hazard ratio (HR) of 7.21 per log unit TLP activity (p = 0.03)]. In contrast, neutrophil elastase displayed no significant associations with lung function or patient survival. Similar findings were evident in the second study cohort. ConclusionsSputum TLP activity may represent a novel non-invasive biomarker and/or therapeutic target for CF lung disease.

Effect of Heterodera schachtii female age on susceptibility to three fungal hyperparasites in the genus Hyalorbilia.

Three closely related nematophagous fungi in the genus Hyalorbilia were compared for their ability to parasitize females and eggs of Heterodera schachtii at different developmental stages. DoUCR50, StM, and ARF were originally isolated from Heterodera schachtii, Meloidogyne incognita, and Heterodera glycines, respectively. Phylogenetic analysis and pairwise sequence analysis showed that DoUCR50 and StM are more closely related to each other than they are to ARF. DoUCR50 parasitism suppressed 100% of the J2 hatch from 3-week-old H. schachtii females and 75% of the hatch from 4-week-old females. Eggs within 5-week-old females were resistant to parasitism, and hatch of J2 was unaffected by exposure to DoUCR50. StM and ARF did not reduce the hatch of J2 from H. schachtii females of any age. Eggs removed from females and spread onto water agar cultures of the fungi were mostly resistant to parasitism. DoUCR50 parasitized only 16% of such eggs from 3-week-old females. Extracellular hydrolytic enzyme production by the three fungal strains grown on PDA or parasitized H. schachtii females was evaluated using API ZYM (bioMérieux) test strips. All three fungi produced extracellular hydrolytic enzymes when grown on PDA or H. schachtii females. Trypsin-like protease activity was uniquely detected in DoUCR50 grown on PDA and H. schachtii females, with the highest activity associated with the fungus grown on parasitized females.

Tryptase: A novel prognostic biomarker in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the presence of the Philadelphia chromosome. It is treated with tyrosine kinase inhibitor agents targeted against the breakpoint cluster region-Abelson murine leukemia 1 fusion transcript. Disease risk stratification at diagnosis of chronic-phase (CP) CML is done using Sokal, Hasford, and EUTOS scores which use basophilia as a major component. However, basophil counts can be both variable and inaccurate. The serum tryptase level is being studied as a novel biomarker which represents the total basophil compartment. Tryptase, deriving its name from trypsin-like activity commonly expressed by mast cells, and also by immature basophils of patients suffering from various myeloid and leukemic disorders, has a role in tumor proliferation. Patients with seemingly low-normal levels of basophils and raised tryptase levels progress further in disease despite treatment. There is a recent interest in the role of serum tryptase as a prognostic marker in CML-CP.

42: Fetuses with fetal growth restriction (FGR) have elevated proteasome levels and activity

To increase lean body mass, the rate of fetal protein synthesis must exceed that of protein breakdown. The ubiquitin-proteasome system is an assembly of enzymatic activities with a key role in protein degradation and homeostasis. Environmental stressors switch the constitutive proteasome (cPROT) to immunoproteasome (iPROT), a quality control proteolytic machinery effective also in the absence of inflammation. Our hypothesis was that FGR fetuses have upregulated levels and enzymatic activity of the proteasome. We analyzed maternal blood, cord blood, and placenta of 70 singleton pregnancies of which 37 were complicated by FGR (EFW< 10%, Table). FGR occurred in the context of preeclampsia with severe features (sPE, n=23), identifiable causes (n=2: CMV infection, true cord knot) or idiopathic factors (n=12). Proteasome levels were measured using ELISA for cPROT and iPROT 20S subunit. Lytic activity was analyzed with fluorogenic substrates for caspase-like (CAS-L), chemotrypsin-like (CHE-L) and trypsin-like (TRY-L) activities. Specificity was confirmed with proteasome inhibitors MG132 and ONX. Placenta was stained for β5 (cPROT) and β5i (iPROT) subunits. Controls were idiopathic preterm births (iPTB) without fetal or placental inflammation. Data was adjusted for gestational age (GA) at birth. 1) The highest maternal cPROT levels were seen in sPE, independent of FGR (sPE p=.003, FGR p=.259, 2-way ANOVA, Fig A); 2) There was no correlation between cord blood and maternal proteasome levels (r=-.009, p=.96); 3) Fetuses with FGR had heightened CHE-L enzymatic activity independent of maternal proteasome levels or activity, sPE status or GA (adjusted p=.04, Fig B); 4) Cord blood proteasome activity of FGR fetuses was inhibited by MG132 and ONX with maximal effect on CHE-L activity which was reduced to 1.7% and 0.6%, respectively; 5) FGR placenta displayed intense β5i staining in trophoblasts and stromal cells suggestive of upregulated iPROT (Fig C). FGR fetuses display a significant upregulation in proteasome enzymatic activity independent of maternal compartment and etiology.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

GPR174 suppression attenuates retinopathy in angiotensin II (Ang II)-treated mice by reducing inflammation via PI3K/AKT signaling

Hypertension is closely associated with eye diseases, including hypertensive retinopathy. However, the molecular mechanism controlling the progression of hypertensive retinopathy remains poorly understood. G-Protein Coupled Receptor 174 (GPR174) is expressed in various tissues, and plays a critical role in regulating immune and inflammatory responses. However, if GPR174 is involved in angiotensin II (Ang II)-induced retinopathy is still unclear. In this study, hypertensive retinopathy was induced by Ang II infusion in the wild type (GPR174+/+) and GPR174 knockout (GPR174−/−) mice. Clinical data indicated that GPR174 levels were markedly up-regulated in serum of patients with hypertension (HP) or hypertensive retinopathy (HR), along with increased trypsin-like activity. Similar changes of GRP174 and trypsin-like activity were observed in Ang II-infused mice. Furthermore, Ang IIsignificantly increased the central retinal thickness, vascular permeability and inflammatory response in GPR174+/+ micewhen compared with the saline GPR174+/+ mice. Of note, these effects were markedly alleviated by the knockout ofGPR174 in Ang II-treated mice. Consistently, vascular endothelial growth factor (VEGF) expression levels in retinal tissue were also stimulated by Ang II, which were clearly attenuated by GPR174−/−. In addition, phosphatidylinositol 3-kinase (PI3 K)/AKT and nuclear factor-κB (NF-κB) signaling pathways were markedly activated in retinas of Ang II-infused GPR174+/+ mice, whereas being greatly ameliorated by GPR174−/−. The in vitro data showed that pre-treatment of PI3 K/AKT specific inhibitor LY294002 remarkably abrogated GPR174 over-expression-accelerated expression levels of Iba-1, tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 in lipopolysaccharide (LPS)-incubated retinal microglial cells.Furthermore, in LPS-exposed retinal microglial cells, PI3 K/AKT and NF-κB pathways were further promoted by GPR174 over-expression, which were significantlyabolished by LY294002. Thus, GPR174 might be a positive meditator of inflammation, contributing to Ang II-induced retinopathy by activating PI3 K/AKT signaling, and could be considered as a novel therapeutic target for the treatment of hypertensive retinopathy.

The effect of dry extract of cabbage garden on the local non-specific proteinase-inhibitory potential of the mucous membrane of the stomach on the model of acute gastric ulcer

Peptic ulcer of the stomach and duodenum is a complex gastroenterological disease multifactorial in pathogenesis. One of the key components of non-specific inflammation in peptic ulcer disease is the activation of proteinases in the gastric mucosa and their interaction with proteinase inhibitors, which determines the degree of proteolytic aggression in terms of the development of destructive changes in the tissues that they damage.&#x0D; The aim of the work was to study the effect of dry extract of cabbage garden on the components of the proteinase-inhibitor system in experimental gastric ulcer.&#x0D; The effect of dry extract of cabbage garden on the local non-specific proteinase-inhibitory potential of the gastric mucosa was studied on the model of acute alcohol-prednisolone ulcers. Dry extract of cabbage garden at a dose of 50 mg/kg and comparison drugs – omeprazole and altan were administered intragastrically once a day for 7 days from the second day of the experiment. On the 8th day, elastase-like, trypsin-like activity and acid-stable inhibitors in the swabs from the stomach and intestines, biopsy samples of the gastric mucosa from the esophagus and fundus, and small intestine were determined.&#x0D; It has been established that with an experimental alcohol-prednisolone gastric ulcer, a local non-specific proteinase-inhibitory system is involved in the mechanism of mucosal damage. At the same time, the level of trypsin-like and elastase-like activity of gastric juice increases and the activity of acid-stable inhibitors decreases, which normally suppress the enzymatic activity of proteinases. According to the effect on the non-specific proteinase-inhibitor system, the dry cabbage extract was significantly superior to altan and not inferior to omeprazole. The most pronounced effect of normalizing the activity of nonspecific proteinases and their inhibitors was established using a combination of dry cabbage extract with omeprazole. One of the mechanisms of the antiulcer action of the dry cabbage extract is its effect on the local nonspecific proteinase-inhibitory potential of the gastric mucosa.&#x0D; In the model of acute alcohol-prednisolone gastric ulcer, dry extract of cabbage garden caused a decrease in the activity of local non-specific proteolytic enzymes and activation of proteinase inhibitors of the gastric mucosa and duodenum, exceeding the activity of altan. Under the combined use of the dry extract of cabbage and omeprazole, a complete normalization of the state of the local non-specific proteinase-inhibitory potential of the gastroduodenal zone was observed.

INDICATORS OF TRYPSIN-LIKE ACTIVITY OF MIXED SALIVAAFTER INSTALLING THE ENDOSSEOUS IMPLANTS IN THE PRESENCE OF GASTRIC ULCER AND DUODENAL ULCER

The trypsin-like activity of mixed saliva in orthopedic patients after implant placement was monitored. Proved negative impact on the processes of repair of background somatic pathology (peptic ulcers and 12 duodenal ulcers), justify the application of immunomodulator "Erbisol" as a drug that speeds up the repair processes during implantation. At present, the relevant issues are those related to the development of inflammatory complications at the stage of rehabilitation of patients, especially in the presence of somatic pathology and the study of the tissue complex of the implantation zone. Peptic ulcer disease occupies one of the main places in the structure of lesions of the digestive system. The close interdependence between the pathology of internal organs and the oral cavity is confirmed by numerous observations and studies.Implant placement is accompanied by changes in the enzymatic activity of mixed saliva. The presence of somatic pathology (ulcerative disease of the stomach and 12 duodenal ulcers) often leads to more long-term violations of the enzymatic activity of saliva. The use of the drug "Erbisol" in orthopedic dentistry accelerates the repair process, as evidenced by the earlier periods of normalization of the enzymatic activity of mixed saliva, after the installation of implants.Regular and timely monitoring of the condition of the periarticular tissues, as well as objective diagnosis of early inflammatory complications, are necessary to ensure the reliability and long-term functioning of prosthetic structures installed on implants in the oral cavity.It can be concluded that the purpose of our study was to study the trypsin-like activity (TPA) of mixed saliva after implant placement on the background of somatic pathology.

The Ubiquitin Proteasome System in Ischemic and Dilated Cardiomyopathy.

Dilated (DCM) and ischemic cardiomyopathies (ICM) are associated with cardiac remodeling, where the ubiquitin–proteasome system (UPS) holds a central role. Little is known about the UPS and its alterations in patients suffering from DCM or ICM. The aim of this study is to characterize the UPS activity in human heart tissue from cardiomyopathy patients. Myocardial tissue from ICM (n = 23), DCM (n = 28), and control (n = 14) patients were used to quantify ubiquitinylated proteins, E3-ubiquitin-ligases muscle-atrophy-F-box (MAFbx)/atrogin-1, muscle-RING-finger-1 (MuRF1), and eukaryotic-translation-initiation-factor-4E (eIF4E), by Western blot. Furthermore, the proteasomal chymotrypsin-like and trypsin-like peptidase activities were determined fluorometrically. Enzyme activity of NAD(P)H oxidase was assessed as an index of reactive oxygen species production. The chymotrypsin- (p = 0.71) and caspase-like proteasomal activity (p = 0.93) was similar between the groups. Trypsin-like proteasomal activity was lower in ICM (0.78 ± 0.11 µU/mg) compared to DCM (1.06 ± 0.08 µU/mg) and control (1.00 ± 0.06 µU/mg; p = 0.06) samples. Decreased ubiquitin expression in both cardiomyopathy groups (ICM vs. control: p < 0.001; DCM vs. control: p < 0.001), as well as less ubiquitin-positive deposits in ICM-damaged tissue (ICM: 4.19% ± 0.60%, control: 6.28% ± 0.40%, p = 0.022), were detected. E3-ligase MuRF1 protein expression (p = 0.62), NADPH-oxidase activity (p = 0.63), and AIF-positive cells (p = 0.50). Statistical trends were detected for reduced MAFbx protein expression in the DCM-group (p = 0.07). Different levels of UPS components, E3 ligases, and UPS activation markers were observed in myocardial tissue from patients affected by DCM and ICM, suggesting differential involvement of the UPS in the underlying pathologies.

Trypanosoma brucei: Inhibition of cathepsin L is sufficient to kill bloodstream forms

The lysosomal cysteine protease activity of Trypanosoma brucei comprises a cathepsin B enzyme (TbCATB) and a cathepsin L enzyme (TbCATL). Inhibition of the cysteine protease activity is lethal to bloodstream-form trypanosomes but it was not entirely clear which of the two enzymes are essential for survival of the parasites. Here we show that the vinyl sulfone compound LU-102 selectively inhibits TbCATL without affecting TbCATB and the proteasomal trypsin-like activity within trypanosomes. Therefore, the trypanocidal activity displayed by LU-102 can be attributed solely to the inhibition of TbCATL demonstrating that this enzyme is essential to the survival of T. brucei.

Traffic-derived air pollution compromises skin barrier function and stratum corneum redox status: A population study.

Since air pollution is only one of many environmental stressors that can affect skin, it has been challenging to identify skin appearance or functional features profoundly affected by chronic exposure to traffic-derived air pollution. The current population study focused on taxi drivers working in urban and rural areas in order to take advantage of difference in occupational exposure. The skin conditions of 100 middle-aged male taxi drivers from urban Shanghai and 66 from rural Chongming were measured with facial tape strips were collected for biomarker analyses. Trans-epidermal water loss (TEWL) values before and after tape stripping were considerably higher in urban taxi drivers from Shanghai. Contrary to previous studies, there was no apparent detrimental effect on skin wrinkle or pigmentation from traffic pollution, which might be attributed to the higher than general public level of photo-exposure in this population. At the same time, pollution exposure especially the heavy traffic pollution exposure was found to associate with lower stratum corneum trypsin-like enzyme activity (SCTE), reduced catalase activity and total antioxidant capacity (TAOC) in tape strips. The evidence suggests that traffic-derived air pollution could deteriorate skin's physical and antioxidant barrier, whereas factors like photo-exposure can be overwhelming against appearance aging. Therefore, in addition to photoprotection, skin barrier care should be considered for people with high air pollution exposure.

Effects of glucose-glycine melanoidins on the digestive trypsin-like activity of the rainbow trout Oncorhynchus mykiss

Melanoidins, a product of advanced browning reactions due to the thermal treatment of the diet, has been previously demonstrated to inhibit the activity of trypsins from mammalian origin, but no data exists in relation to fish trypsins. The present work is devoted to the investigation of the effects of glucose-glycine melanoidins on the trypsin-like activity contained in intestinal crude extracts (ECIs) prepared from juvenile individuals of the rainbow trout Oncorhynchus mykiss. A turbidity assay was conducted to test for the existence of precipitation or aggregation phenomena in mixtures of ECIs and melanoidins, but no consequences were observed. Three experiments were conducted to test for the effects on the trypsin-like activity on the hydrolysis of the substrate BAEE (Nα-Benzoyl-L-arginine ethyl ester). Glucose-glycine melanoidins can inhibit the intestinal trypsin-like activity of the rainbow trout, but the effect is modulated by the pH and ionic strength of the reaction medium. The inhibition dropped when the pH was changed from 7.5 to 8.5, also when the ionic strength of the buffer was raised from 40 to 140 mM, while it was undetected at 180 mM. It is concluded that the effect of this type of melanoidins on the trypsin-like activity from the rainbow trout is probably low in the ionic conditions prevailing in the intestine of this species.

Ochratoxin A Sequentially Activates Autophagy and the Ubiquitin-Proteasome System.

Ochratoxin A (OTA) is a carcinogenic mycotoxin, which is produced by Aspergillus and Penicillium genera of fungi and commonly contaminates food and feed. We and others have previously shown that OTA causes sustained activation of PI3K/AKT and MAPK/ERK1-2 signaling pathways in different cell types and animal models. Given the close relationship between cellular signaling activity and protein stability, we were curious whether increased PI3K/AKT and MAPK/ERK1-2 signaling may be the result of OTA-stimulated alterations in proteolytic activity. We show that both of the major proteolytic systems, autophagy, and the ubiquitin-proteasome system (UPS), are activated upon OTA exposure in human kidney proximal tubule HK-2 and mouse embryonic fibroblast (MEF) cells. OTA stimulates transient autophagic activity at early time points of treatment but autophagic activity subsides after 6 h even in the sustained presence of OTA. Interestingly, OTA exposure also results in increased cell death in wild-type MEF cells but not in autophagy-halted Atg5-deficient cells, suggesting that autophagy exerts a pro-death effect on OTA-induced cytotoxicity. In addition, prolonged OTA exposure decreased ubiquitinated protein levels by increasing proteasomal activity. Using purified and cellular proteasomes, we observed enhanced chymotrypsin-, caspase-, and trypsin-like activities of the 26S but not the 20S proteasome in the presence of OTA. However, in the cellular context, increased proteasomal activity depended on prior induction of autophagy. Our results suggest that autophagy and subsequent UPS activation are responsible for sustained activation of PI3K/AKT and MAPK/ERK1-2 pathways through regulating the levels of critical phosphatases VHR/DUSP3, DUSP4, and PHLPP, which are known to be involved in OTA toxicity and carcinogenicity.

Larval development and proteolytic activity of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) exposed to different soybean protease inhibitors.

Anticarsia gemmatalis represents a relevant factor for lowering soybean and other legumecropproductivities. Protease inhibitors affectprotein degradation and reducethe availability of amino acids, impairing the development and survival of insect pests. To evaluate the possible use of proteinaceous protease inhibitors in the management of this pest, the activities of midgut proteases andthe growth and development of A. gemmatalis larvae exposed to soybean Bowman-Birk trypsin-chymotrypsin inhibitor (SBBI) and soybean Kunitz trypsin inhibitor (SKTI) were determined. The survival curves obtained using Kaplan-Meier estimators indicated that SKTI and SBBI stimulated larval survival. However, the development of A. gemmataliswas delayed, and prepupal weight decreased in the presence of both inhibitors. The results showed that SKTI and SBBI inhibited the trypsin-like and total proteolytic activities of larvae on the 12th day after eclosion. On the 15th day after eclosion, larvae exposed to SKTI increased the activities of trypsin and total proteases. Although SKTI and SBBI did not affect the survival of the insect, they had effects on midgut proteases in a stage whereinA. gemmatalis fedvoraciously, increased the larval cycle, and decreased prepupal weight. These findings provide baseline informationabout the potential of proteinaceous protease inhibitors to manage the velvetbean caterpillar, avoiding chemical pesticides.

Effects of the serine protease inhibitor rBmTI-A in an experimental mouse model of chronic allergic pulmonary inflammation

To evaluate whether a recombinant serine protease inhibitor (rBmTI-A) modulates inflammation in an experimental model of chronic allergic lung inflammation. Balb/c mice were divided into four groups: SAL (saline), OVA (sensitized with ovalbumin), SAL + rBmTI-A (control treated with rBmTI-A) and OVA + rBmTI-A (sensitized with ovalbumin and treated with rBmTI-A). The animals received an intraperitoneal injection of saline or ovalbumin, according to the group. The groups received inhalation with saline or ovalbumin and were treated with rBmTI-A or saline by nasal instillation. After 29 days, we evaluated the respiratory mechanics; bronchoalveolar lavage fluid (BALF); cytokines; MMP-9, TIMP-1; eosinophils; collagen and elastic fibre expression in the airways; and the trypsin-like, MMP-1, and MMP-9 lung tissue proteolytic activity. Treatment with rBmTI-A reduced the trypsin-like proteolytic activity, the elastance and resistance maximum response, the polymorphonuclear cells, IL-5, IL-10, IL-13 and IL-17A in the BALF, the expression of IL-5, IL-13, IL-17, CD4+, MMP-9, TIMP-1, eosinophils, collagen and elastic fibres in the airways of the OVA + rBmTI-A group compared to the OVA group (p < 0.05). rBmTI-A attenuated bronchial hyperresponsiveness, inflammation and remodelling in this experimental model of chronic allergic pulmonary inflammation. This inhibitor may serve as a potential therapeutic tool for asthma treatment.

Measuring redox effects on the activities of intracellular proteases such as the 20S Proteasome and the Immuno-Proteasome with fluorogenic peptides

Proteolytic enzymes are often strongly affected by redox reactions, free radicals, oxidation, or oxidative stress. The 20S Proteasome and the Immuno-Proteasome are examples of major intracellular proteases whose concentration, transcription, translation, and proteolytic activity are all subject to redox regulation. Proteasomes are essential in maintaining overall protein homeostasis (or proteostasis), and their dysregulation results in detrimental phenotypes associated with various pathologies, including several common age-related diseases. Many studies have used Western blots to assess redox changes in Proteasome protein levels or RT-PCR to study RNA transcript levels, but actual measurements of proteolytic activity are far less common. Since each intact protein substrate exhibits a different proteolytic profile when incubated with proteasome or Immuno-Proteasome [± activators such as 19S or 11S (also called PA28)] and these proteolytic profiles are drastically altered if the protein substrate is denatured, for example by oxidation, heat, acetylation, or methylation. In an attempt to standardize proteasomal activity measurements small fluorogenic protein/peptide substrates were developed to test the three proteolytically active sites of the Proteasome and Immuno-Proteasome: trypsin-like, chymotrypsin-like, and caspase-like activities. Despite extensive use of fluorogenic peptide substrates to measure proteasome activity, there is an absence of a standardized set of best practices. In this study we analyze different parameters, such as sample concentration, AMC conjugated substrate concentration, duration of assay, and frequency of measurements, and examine how they impact the determination of Proteasome and Immuno-Proteasome activities using fluorogenic peptide substrates.