Proteolytic Responses Research Articles

Gelatinolytic activity after dentin pretreatment with dimethyl sulfoxide (DMSO) combined to dental bonding systems: Perspectives for biological responses

Endogeneous proteolytic responses in dentin bonding interface have addressing to strategies to preventive and therapeutic approaches of clinical use of dentin bonding systems (DBSs), but still present limitations. The aim of this study was to examine the gelatinolytic profile by means of in situ zymography regarding the use of 1% dimethyl sulfoxide (DMSO) as an aprotic solvent. Sound human third molars were prepared and randomized in 10 groups, following the factors 1- DBS: Adper™ Scotchbond Multipurpose [MP], Adper™ Single Bond 2 [SB], Clearfil™ SE Bond [CSE] and Adper™ Scotchbond Universal – Etch-and-rinse [SU-ER] mode and self-etch mode [SU-SE], 2- dentin pretreatment: Control – Water [C], 2% CHX and 1% DMSO and 3- time: Initial-24 h [I], 6 months [6M] and 30 months [30M]. Pretreatments were applied before primer application for 30s. After restoration, specimens were cut into slices, in which one third were incubated with fluorescein-conjugated gelatin for 24h at 37 °C and analyzed by confocal laser scanning microscopy. The other two-thirds were stored for 6 or 30 months at 37 °C. Fluorescence was quantified using Image J and data was subjected for two-way ANOVA followed by Tukey test (p<0.05). Neither DMSO nor CHX affected initial analyses for any tested conditions. After 6 months, it was observed increased fluorescence for MP using both pretreatments and for SB using only DMSO. Regardless time and pretreatment, CSE and SU-SE showed stabilized gelatinolytic pattern. For SU-ER, both CHX and DMSO were able to maintain a lower fluorescence compared to control group after 6 months. 30-month performance states the susceptibility of degradation for all etched-dentin systems. DMSO pretreatment can be promising to reduce gelatinolytic activity combined with an universal adhesive system under etch-and-rinse mode. For self-etching strategies, DMSO was successful to stabilize the gelatinolytic reactions.

Effects of the Fungal Bioherbicide, Alternaria cassia on Peroxidase, Pectinolytic and Proteolytic Activities in Sicklepod Seedlings.

Certain plant pathogens have demonstrated potential for use as bioherbicides for weed control, and numerous studies have been published on this subject for several decades. One of the early examples of an important fungal bioherbicide is Alternaria cassiae, isolated from the weed sicklepod (Senna obtusifolia). To gain further insight into biochemical interactions of this fungus and its host weed, we examined the effects of this bioherbicide on various enzymes associated with plant defense. Young sicklepod seedlings were challenged with A. cassiae spore inoculum and enzyme activities associated with plant defense (peroxidase, proteolytic, and pectinolytic) were assayed periodically over a 96-h time course on plants grown in continuous darkness or continuous light. Peroxidase activity increased with time in untreated control seedlings in both light and dark, but the effect was greater in the light. In A. cassiae-treated plants, peroxidase was elevated above that in control tissue at all sample times resulting in a 1.5 -fold increase above control in light-grown tissue and a 2- to 3-fold increase in dark-grown tissue over 48–96 h. Differences in leucine aminopeptidase activity in control versus A. cassiae-treated tissues were not significant until 48–96 h, when activity was inhibited in fungus-treated tissues by about 32% in light-grown tissue and 27% in dark-grown tissue after 96 h. Proteolytic activity on benzoyl-arginine-p-nitroanilide was not significantly different in treated versus control tissue in either light or dark over the time course. Pectinase activity increased in treated tissues at all time points as early as 16 h after spore application in light- or dark-grown plants. The greatest increases were 1.5-fold above control levels in light-grown plants (40–64 h) and 2-fold in plants grown in darkness (72–96 h). Data suggests that peroxidase may be involved as defense mechanism of sicklepod when challenged by A. cassia and that this mechanism is operative in young seedlings under both light and dark growth conditions. Differential proteolytic activity responses on these two substrates suggests the presence of two different enzymes. Increased pectinase activity during pathogenesis suggests that A. cassiae-sicklepod interaction results in an infectivity mechanism to degrade pectic polymers important to sicklepod cell wall integrity. These studies provide important information on some biochemical interactions that may be useful for improvements to biological weed control programs utilizing plant pathogens. Such information may also be useful in genetic selection and manipulation of pathogens for weed control.

Chronic Cigarette Smoke Exposure Subdues PP2A Activity by Enhancing Expression of the Oncogene CIP2A.

Phosphatase activity of the major serine threonine phosphatase, protein phosphatase 2A (PP2A), is blunted in the airways of individuals with chronic obstructive pulmonary disease (COPD), which results in heightened inflammation and proteolytic responses. The objective of this study was to investigate how PP2A activity is modulated in COPD airways. PP2A activity and endogenous inhibitors of PP2A were investigated in animal and cell models of COPD. In primary human bronchial epithelial (HBE) cells isolated from smokers and donors with COPD, we observed enhanced expression of cancerous inhibitor of PP2A (CIP2A), an oncoprotein encoded by the KIAA1524 gene, compared with cells from nonsmokers. CIP2A expression was induced by chronic cigarette smoke exposure in mice that coincided with a reduction in PP2A activity, airspace enlargements, and loss of lung function, as determined by PP2A phosphatase activity, mean linear intercept analysis, and forced expiratory volume in 0.05 second/forced vital capacity. Modulating CIP2A expression in HBE cells by silencing RNA or chemically with erlotinib enhanced PP2A activity, reduced extracellular-signal-regulated kinase phosphorylation, and reduced the responses of matrix metalloproteinases 1 and 9 in HBE cells isolated from subjects with COPD. Enhanced epithelial growth factor receptor responses in cells from subjects with COPD were observed to modulate CIP2A expression levels. Our study indicates that chronic cigarette smoke induction of epithelial growth factor receptor signaling and CIP2A expression can impair PP2A responses that are associated with loss of lung function and enhancement of proteolytic responses. Augmenting PP2A activity by manipulating CIP2A expression may represent a feasible therapeutic approach to counter smoke-induced lung disease.

PI3K-AKT-FOXO1 pathway targeted by skeletal muscle microRNA to suppress proteolytic gene expression in response to carbohydrate intake during aerobic exercise.

Ingesting protein and carbohydrate together during aerobic exercise suppresses the expression of specific skeletal muscle microRNA and promotes muscle hypertrophy. Determining whether there are independent effects of carbohydrate and protein on microRNA will allow for a clearer understanding of the mechanistic role microRNA serve in regulating skeletal muscle protein synthetic and proteolytic responses to nutrition and exercise. This study determined skeletal muscle microRNA responses to aerobic exercise with or without carbohydrate, and recovery whey protein (WP). Seventeen males were randomized to consume carbohydrate (CHO; 145 g; n = 9) or non‐nutritive control (CON; n = 8) beverages during exercise. Muscle was collected before (BASE) and after 80 min of steady‐state exercise (1.7 ± 0.3 V̇O2 L·min−1) followed by a 2‐mile time trial (17.9 ± 3.5 min; POST), and 3‐h into recovery after consuming WP (25 g; REC). RT‐qPCR was used to determine microRNA and mRNA expression. Bioinformatics analysis was conducted using the mirPath software. Western blotting was used to assess protein signaling. The expression of six microRNA (miR‐19b‐3p, miR‐99a‐5p, miR‐100‐5p, miR‐222‐3p, miR‐324‐3p, and miR‐486‐5p) were higher (P < 0.05) in CHO compared to CON, all of which target the PI3K‐AKT, ubiquitin proteasome, FOXO, and mTORC1 pathways. p‐AKTT hr473 and p‐FOXO1Thr24 were higher (P < 0.05) in POST CHO compared to CON. The expression of PTEN was lower (P < 0.05) in REC CHO than CON, while MURF1 was lower (P < 0.05) POST CHO than CON. These findings suggest the mechanism by which microRNA facilitate skeletal muscle adaptations in response to exercise with carbohydrate and protein feeding is by inhibiting markers of proteolysis.

Effect Of Repeated Bouts Of Resistance Exercise On Skeletal Muscle Proteolytic Response In Rat.

Resistance training (RT) increases the skeletal muscle mass and strength. It has been reported that activation of the mammalian target of rapamycin (mTOR) signaling, which is important for skeletal muscle protein synthesis, is attenuated by increasing exercise bout. However, the effect on proteolytic response is unclear. PURPOSE: The present study aimed to investigate the changes in proteolytic responses to repeated bouts of resistance exercised. METHODS: Male Sprague-Dawley rats were randomly assigned into four groups: Sedentary (SED), resistance-exercised with 1bout (1B), 2bouts (2B), 3bouts (3B). RT protocol consisted of 50 repetitions of maximal isometric contraction of the right gastrocnemius muscle by direct electric stimulation under anesthesia. Muscle samples were collected 3h and 6h after the final exercise session. RESULTS: Phosphorylation of p70S6K increased in all trained groups (1B: 15 fold, 2B: 10 fold, 3B: 8 fold, P<0.05). And there was also significant difference between 1B and 3B. The protein ubiquitination increased in all trained groups of 1.2-fold in SED (P<0.05). And Light chain 3 (LC3), which is a marker for autophagy, increased in all trained groups of 1.5-fold in SED (P<0.05). MuRF-1 mRNA showed a significant 5.1-fold increase with training (P<0.05). However, there were no effect of repeated bouts of RT in muscle proteolytic response. CONCLUSIONS: The present results suggest that the muscle protein proteolytic response was activated by RT. In addition, the activation of mTOR signaling attenuated with the increase in RT bouts, but the level of activation of proteolytic response did not change.

Aging attenuates redox adaptive homeostasis and proteostasis in female mice exposed to traffic-derived nanoparticles ('vehicular smog').

Environmental toxicants are catalysts for protein damage, aggregation, and the aging process. Fortunately, evolution selected adaptive homeostasis as a system to mitigate such damage by expanding the normal capacity to cope with toxic stresses. Little is known about the subcellular degradative responses to proteins oxidatively damaged by air pollution. To better understand the impact of environmental toxicants upon the adaptive homeostatic response, female C57BL/6 mice were exposed for 10 weeks to filtered air or reaerosolized vehicular-derived nano-scale particulate matter (nPM), at which point tissues from young (6 month) and middle-aged (21 month) mice were studied. We found significant increases of proteolytic capacity in lung, liver, and heart. Up to two-fold increases were seen in the 20S Proteasome, the Immunoproteasome, the mitochondrial Lon protease, and NF-E2-related factor 2 (Nrf2), a major transcriptional factor for these and other stress-responsive genes. The responses were equivalent in all organs, despite the indirect input of inhaled particles to heart and liver which are downstream of lung. To our knowledge, this is the first exploration of proteostatic responses to oxidative damage by air pollution. Although, middle-aged mice had higher basal levels, their Nrf2-responsive-genes exhibited no response to nanoparticulate exposure. We also found a parallel age-associated rise in the Nrf2 transcriptional inhibitors, Bach1 and c-Myc which appear to attenuate adaptive responses in older mammals, possibly explaining the 'age-ceiling effect.' This report extends prior findings in male mice by demonstrating the involvement of proteolytic responses to traffic-related air pollution in lung, liver, and heart of female mice, with an age-dependent loss of adaptive homeostasis.

The effect of different acute muscle contraction regimens on the expression of muscle proteolytic signaling proteins and genes.

Previous studies have reported that different modes of muscle contraction (i.e., eccentric or concentric contraction) with similar contraction times can affect muscle proteolytic responses. However, the effect of different contraction modes on muscle proteolytic response under the same force−time integral (FTI: contraction force × time) has not been investigated. The purpose of this study was to investigate the effect of different contraction modes, with the same FTI, on acute proteolytic signaling responses. Eleven‐week‐old male Sprague–Dawley rats were randomly assigned to eccentric (EC), concentric (CC), or isometric contraction (IC) groups. Different modes of muscle contraction were performed on the right gastrocnemius muscle using electrical stimulation, with the left muscle acting as a control. In order to apply an equivalent FTI, the number of stimulation sets was modified between the groups. Muscle samples were taken immediately and three hours after exercise. Phosphorylation of FoxO3a at Ser253 was significantly increased immediately after exercise compared to controls irrespective of contraction mode. The mRNA levels of the ubiquitin ligases, MuRF1, and MAFbx mRNA were unchanged by contraction mode or time. Phosphorylation of ULK1 at Ser317 (positive regulatory site) and Ser757 (negative regulatory site) was significantly increased compared to controls, immediately or 3 h after exercise, in all contraction modes. The autophagy markers (LC3B‐II/I ratio and p62 expression) were unchanged, regardless of contraction mode. These data suggest that differences in contraction mode during resistance exercise with a constant FTI, are not factors in regulating proteolytic signaling in the early phase of skeletal muscle contraction.

Inhibitors of ceramide de novo biosynthesis rescue damages induced by cigarette smoke in airways epithelia.

Exposure to cigarette smoke represents the most important risk factor for the development of chronic obstructive pulmonary disease (COPD). COPD is characterized by chronic inflammation of the airways, imbalance of proteolytic activity resulting in the destruction of lung parenchyma, alveolar hypoxia, oxidative stress, and apoptosis. Sphingolipids are structural membrane components whose metabolism is altered during stress. Known as apoptosis and inflammation inducer, the sphingolipid ceramide was found to accumulate in COPD airways and its plasma concentration increased as well. The present study investigates the role of sphingolipids in the cigarette smoke-induced damage of human airway epithelial cells. Lung epithelial cells were pre-treated with sphingolipid synthesis inhibitors (myriocin or XM462) and then exposed to a mixture of nicotine, acrolein, formaldehyde, and acetaldehyde, the major toxic cigarette smoke components. The inflammatory and proteolytic responses were investigated by analysis of the mRNA expression (RT-PCR) of cytokines IL-1β and IL-8, and matrix metalloproteinase-9 and of the protein expression (ELISA) of IL-8. Ceramide intracellular amounts were measured by LC-MS technique. Ferric-reducing antioxidant power test and superoxide anion radical scavenging activity assay were used to assess the antioxidant power of the inhibitors of ceramide synthesis. We here show that ceramide synthesis is enhanced under treatment with a cigarette smoke mixture correlating with increased expression of inflammatory cytokines and matrix metalloproteinase 9. The use of inhibitors of ceramide synthesis protected from smoke induced damages such as inflammation, oxidative stress, and proteolytic imbalance in airways epithelia.

Plasminogen activator inhibitor-1 regulates macrophage-dependent postoperative adhesion by enhancing EGF-HER1 signaling in mice.

Adhesive small bowel obstruction remains a common problem for surgeons. After surgery, platelet aggregation contributes to coagulation cascade and fibrin clot formation. With clotting, fibrin degradation is simultaneously enhanced, driven by tissue plasminogen activator-mediated cleavage of plasminogen to form plasmin. The aim of this study was to investigate the cellular events and proteolytic responses that surround plasminogen activator inhibitor (PAI-1; Serpine1) inhibition of postoperative adhesion. Peritoneal adhesion was induced by gauze deposition in the abdominal cavity in C57BL/6 mice and those that were deficient in fibrinolytic factors, such as Plat-/- and Serpine1-/- In addition, C57BL/6 mice were treated with the novel PAI-1 inhibitor, TM5275. Some animals were treated with clodronate to deplete macrophages. Epidermal growth factor (EGF) experiments were performed to understand the role of macrophages and how EGF contributes to adhesion. In the early phase of adhesive small bowel obstruction, increased PAI-1 activity was observed in the peritoneal cavity. Genetic and pharmacologic PAI-1 inhibition prevented progression of adhesion and increased circulating plasmin. Whereas Serpine1-/- mice showed intra-abdominal bleeding, mice that were treated with TM5275 did not. Mechanistically, PAI-1, in combination with tissue plasminogen activator, served as a chemoattractant for macrophages that, in turn, secreted EGF and up-regulated the receptor, HER1, on peritoneal mesothelial cells, which led to PAI-1 secretion, further fueling the vicious cycle of impaired fibrinolysis at the adhesive site. Controlled inhibition of PAI-1 not only enhanced activation of the fibrinolytic system, but also prevented recruitment of EGF-secreting macrophages. Pharmacologic PAI-1 inhibition ameliorated adhesion formation in a macrophage-dependent manner.-Honjo, K., Munakata, S., Tashiro, Y., Salama, Y., Shimazu, H., Eiamboonsert, S., Dhahri, D., Ichimura, A., Dan, T., Miyata, T., Takeda, K., Sakamoto, K., Hattori, K., Heissig, B. Plasminogen activator inhibitor-1 regulates macrophage-dependent postoperative adhesion by enhancing EGF-HER1 signaling in mice.

Aluminum induces inflammatory and proteolytic alterations in human monocytic cell line

The increasing exposure to aluminum has been linked with the development of different human pathologies (e.g., breast cancer, myofasciitis, neurodegenerative diseases), probably due to the consistent presence of aluminum salts in widely diffused cosmetic products and vaccines. However, the mechanisms underlying immunologic and proliferative alterations still remain unknown.In the present study we investigated the ability of different aluminum compounds (i.e., aluminum chloride vs Imject® Alum, a mixture of aluminum and magnesium hydroxide) to trigger both inflammatory and proteolytic responses in U-937 human monocytic cell line. We demonstrated, by multiplex immunoassay analyses, that monocytic cells treated with both Imject Alum and aluminum chloride showed different and peculiar expression profiles of 27 inflammatory mediators and 5 matrix metalloproteinases, with respect to untreated control cells.In particular, we found dose-dependent significantly increased levels of pro-inflammatory cytokines, growth factors, and chemoattractant chemokines; whereas among metalloproteinases, only collagenolytic protease showed a significant dose-dependent increase in Imject-treated cells with respect to controls and Al-chloride treated cells. Noteworthy, we found only in Imject Alum-treated cells the significant positive correlations among collagenolytic metalloproteinase and increased expression of pro-inflammatory chemokines, suggesting a possible involvement of aluminum in regulating the acute inflammatory responses.In agreement to emerging evidences, for the first time we demonstrated that the treatment of monocyte cells with aluminum-based adjuvant is able to induce an inflammatory status and a proteolytic cascade activation. In fact, the cell treatment with Imject Alum induced increased levels of several cytokines and proteinases, suggesting these monocyte mediators as possible biomarkers for aluminum-linked diseases. The identification of the biochemical pathways involved in Al-induced cell injury pave the way for improving the knowledge on the potential impact of aluminum in human physio-pathology.

α1-Antitrypsin activates protein phosphatase 2A to counter lung inflammatory responses.

α1-Antitrypsin (A1AT) was identified as a plasma protease inhibitor; however, it is now recognized as a multifunctional protein that modulates immunity, inflammation, proteostasis, apoptosis, and cellular senescence. Like A1AT, protein phosphatase 2A (PP2A), a major serine-threonine phosphatase, regulates similar biologic processes and plays a key role in chronic obstructive pulmonary disease. Given their common effects, this study investigated whether A1AT acts via PP2A to alter tumor necrosis factor (TNF) signaling, inflammation, and proteolytic responses in this disease. PP2A activity was measured in peripheral blood neutrophils from A1AT-deficient (PiZZ) and healthy (PiMM) individuals and in alveolar macrophages from normal (60 mg/kg) and high-dose (120 mg/kg) A1AT-treated PiZZ subjects. PP2A activation was assessed in human neutrophils, airway epithelial cells, and peripheral blood monocytes treated with plasma purified A1AT protein. Similarly, lung PP2A activity was measured in mice administered intranasal A1AT. PP2A was silenced in lung epithelial cells treated with A1AT and matrix metalloproteinase and cytokine production was then measured following TNF-α stimulation. PP2A was significantly lower in neutrophils isolated from PiZZ compared with PiMM subjects. A1AT protein activated PP2A in human alveolar macrophages, monocytes, neutrophils, airway epithelial cells, and in mouse lungs. This activation required functionally active A1AT protein and protein tyrosine phosphatase 1B expression. A1AT treatment acted via PP2A to prevent p38 and IκBα phosphorylation and matrix metalloproteinase and cytokine induction in TNF-α-stimulated epithelial cells. Together, these data indicate that A1AT modulates PP2A to counter inflammatory and proteolytic responses induced by TNF signaling in the lung.

Responses of proteolytic enzymes in embryonic axes of germinating bean seeds under copper stress.

The changes in protease activities in embryonic axes during the first days of bean (Phaseolus vulgaris L.) seed germination were investigated in response to copper stress. Synthetic substrates and specific protease inhibitors have been used to define qualitatively and quantitatively different catalytic classes, particularly endoproteases (EP), carboxypeptidases (CP) and aminopeptidases (AP), then identify which ones were affected in the presence of copper. In fact, a failure in storage proteins mobilization and a disorder of nitrogen supply at enzymatic level occurred in Cu. In fact, Cu inhibited azocaseinolytic activity (ACA) and cysteine-, aspartic-, serine-, and metallo-endopeptidases activities (Cys-EP, Asp-EP, Ser-Ep, and Met-EP, respectively). Besides, Cu affected leucine- and proline-aminopeptidases (LAP and PAP, respectively) and glycine-carboxypeptidases (Gly-CP). The proteolytic responses might also be associated with the decrease in defense capacity in the Cu-treated embryos.

Signaling control of proteasome, immunoproteasome, and PA28 regulator gene expression during adaptation to oxidative stress (555.11)

Levels of oxidative stress change continuously, depending on environment, metabolism, foods, drugs and medications, and radiation. Young, healthy, mammalian cells can readily adapt to increases in oxidative stress such that they become (temporarily) much more resistant to oxidative damage. Intracellular proteins are major targets of oxidative stress and it is vitally important that cells rapidly and selectively degrade mildly oxidized proteins, before they undergo more severe oxidation, aggregation, and cross‐linking. Our studies show that such adaptive responses include the immediate disassembly of the 26S proteasome (catalyzed by HSP70 and Ecm‐29) to form free 20S proteasome and 19S regulator complexes, at which point ATP/Ubiquitin‐dependent proteolysis is temporarily lost. The additional free 20S Proteasomes (+/‐ 11S or PA28 regulators) are of immediate help in degrading oxidized proteins. The original 26S Proteasomes are re‐assembled, and ATP/Ubiquitin‐dependent proteolysis is restored over a three‐hour period. During this three‐hour period, and during the subsequent 17 hours, new 20S Proteasomes, Immunoproteasomes, and 11S (PA28) proteasome regulators are synthesized, partially under the control of the Nrf2 signal transduction pathway. We have now obtained similar findings in C. elegans worms, and D. melanogaster fruit flies. Proteasome and Immunoproteasome subunit genes, and the 11S (PA28) regulator appear to be true shock/stress genes that provide significant oxidative stress protection. In older cells, Proteasome activities decline, and adaptational responses mediated by Nrf2 become sluggish or even ineffectual. The antagonistic effects of Bach1 and/or Nrf1 may actually blunt adaptive proteolytic responses to stress in older cells and organisms. Studies in animals and humans suggest that declining Proteasome activities and, perhaps, declining responsiveness to stress, may contribute to the ageing process, and to various age‐associated diseases.Grant Funding Source: NIH/NIEHS ES003598

The Glutathione Peroxidase 1–Protein Tyrosine Phosphatase 1B–Protein Phosphatase 2A Axis. A Key Determinant of Airway Inflammation and Alveolar Destruction

Protein phosphatase-2A (PP2A) is a primary serine-threonine phosphatase that modulates inflammatory responses in asthma and chronic obstructive pulmonary disease (COPD). Despite its importance, the mechanisms that regulate lung PP2A activity remain to be determined. The redox-sensitive enzyme protein tyrosine phosphatase-1B (PTP1B) activates PP2A by dephosphorylating the catalytic subunit of the protein at tyrosine 307. This study aimed to identify how the interaction between the intracellular antioxidant glutathione peroxidase-1 (GPx-1) and PTP1B affected lung PP2A activity and airway inflammation. Experiments using gene silencing techniques in mouse lung or human small airway epithelial cells determined that knocking down PTP1B expression blocked GPx-1's activation of PP2A and negated the anti-inflammatory effects of GPx-1 protein in the lung. Similarly, the expression of human GPx-1 in transgenic mice significantly increased PP2A and PTP1B activities and prevented chronic cigarette smoke-induced airway inflammation and alveolar destruction. GPx-1 knockout mice, however, exhibited an exaggerated emphysema phenotype, correlating with a nonresponsive PP2A pathway. Importantly, GPx-1-PTP1B-PP2A signaling becomes inactivated in advanced lung disease. Indeed, PTP1B protein was oxidized in the lungs of subjects with advanced emphysema, and cigarette smoke did not increase GPx-1 or PTP1B activity within epithelial cells isolated from subjects with COPD, unlike samples of healthy lung epithelial cells. In conclusion, these findings establish that the GPx-1-PTP1B-PP2A axis plays a critical role in countering the inflammatory and proteolytic responses that result in lung-tissue destruction in response to cigarette smoke exposure.

A Comparison of the Inflammatory and Proteolytic Effects of Dung Biomass and Cigarette Smoke Exposure in the Lung

RationaleBiomass is the energy source for cooking and heating for billions of people worldwide. Despite their prevalent use and their potential impact on global health, the effects of these fuels on lung biology and function remain poorly understood.MethodsWe exposed human small airway epithelial cells and C57BL/6 mice to dung biomass smoke or cigarette smoke to compare how these exposures impacted lung signaling and inflammatory and proteolytic responses that have been linked with disease pathogenesis.ResultsThe in vitro exposure and siRNA studies demonstrated that biomass and cigarette smoke activated ERK to up regulate IL-8 and MMP-1 expression in human airway epithelial cells. In contrast to cigarette smoke, biomass also activated p38 and JNK within these lung cells and lowered the expression of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1). Similarly, in the lungs of mice, both biomass and cigarette smoke exposure increased macrophages, activated ERK and p38 and up regulated MMP-9 and MMP-12 expression. The main differences seen in the exposure studies was that mice exposed to biomass exhibited more perivascular inflammation and had higher G-CSF and GM-CSF lavage fluid levels than mice exposed identically to cigarette smoke.ConclusionBiomass activates similar pathogenic processes seen in cigarette smoke exposure that are known to result in the disruption of lung structure. These findings provide biological evidence that public health interventions are needed to address the harm associated with the use of this fuel source.

Compensatory proteolytic responses to dietary proteinase inhibitors from Albizia lebbeck seeds in the Helicoverpa armigera larvae

Plant proteinase inhibitors (PIs) have been shown to reduce the growth rates in larvae of numerous insect species. On the other hand, insects can also regulate their proteinases against plant PIs. In the present study, we report the compensatory activities of Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) gut proteinases against the PIs of Albizia lebbeck seeds. Total of ten proteinase inhibitor bands were detected in the seed extract of A. lebbeck. Bioassays were conducted by feeding H. armigera larvae on diet containing partially purified PIs from A. lebbeck seeds. Results show that larval growth and survival was significantly reduced by A. lebbeck PIs. We found that higher activity H. armigera gut proteinase (HGP) isoforms observed in the midgut of control larvae were inhibited in the midgut of larvae fed on test diet. Some HGP isoforms were induced in the larvae fed on PI containing test diet; however, these isoforms showed lower activity in the larvae fed on control diet. Aminopeptidase activities were significantly increased in the midgut of larvae fed on test diet. A population of susceptible and resistant enzymes was observed in the midgut of H. armigera, when fed on diet containing PIs from A. lebbeck seeds. Our initial observations indicate that H. armigera can regulate its digestive proteinase activity against non-host plant PIs, too. It is important to study the exact biochemical and molecular mechanisms underlying this phenomenon in order to develop PI-based insect control strategies.

Effects of lysophosphatidic acid on calpain‐mediated proteolysis of focal adhesion kinase in human prostate cancer cells

Calcium-mediated proteolysis plays an important role in cell migration. Lysophosphatidic acid (LPA), a lipid mediator present in serum, enhances migration of carcinoma cells. The effects of LPA on calpain-mediated proteolysis were, therefore, examined in PC-3, a human prostate cancer cell line. Cultured PC-3 cells were used in studies utilizing pharmacologic interventions, immunoblotting, and confocal immunolocalization. Focal adhesion kinase (FAK), a tyrosine kinase involved in cell adhesion, is rapidly proteolyzed in serum-starved PC-3 cells exposed to the calcium ionophore, ionomycin; Nck, p130CAS, PKCα, and Ras-GAP are also degraded. Thapsigargin, which causes more moderate increases in intracellular calcium, induces partial proteolysis of these proteins. Calpain inhibitors block the proteolytic responses to ionomycin and thapsigargin. Ionomycin does not induce proteolysis in cells maintained in serum, suggesting a protective role for growth factors contained in serum. LPA causes minor FAK proteolysis when added alone, but protects against ionomycin-induced proteolysis in a time-dependent manner. LPA also protects against the cell detachment that eventually follows ionomycin treatment. The response to LPA is blocked by an LPA receptor antagonist. A similar effect of LPA is observed in ionomycin-treated Rat-1 fibroblasts. In PC-3 cells, the protective effects of LPA and serum are correlated with phosphorylation and redistribution of paxillin, suggesting roles for phosphorylation-mediated protein-protein interactions. The complex effects of LPA on calpain-mediated proteolysis of FAK and other adhesion proteins are likely to play a role in the ability of LPA to promote attachment, migration, and survival of prostate cancer cells.

Protein Phosphatase 2A Regulates Innate Immune and Proteolytic Responses to Cigarette Smoke Exposure in the Lung

Protein phosphatase 2A (PP2A) is the primary serine-threonine phosphatase of eukaryotic cells, and changes in its activity have been linked to neoplastic and neurodegenerative diseases. However, the role of PP2A in noncancerous lung diseases such as chronic obstructive pulmonary disease (COPD) has not been previously examined. This study determined that PP2A activity was significantly increased in the lungs of advanced emphysema subjects compared with age-matched controls. Furthermore, we found that cigarette smoke exposure increases PP2A activity in mouse lung in vivo and in primary human small airway epithelial (SAE) cells in vitro. In mice, intratracheal transfection of PP2A protein prior to cigarette smoke exposure prevented acute smoke-induced lung inflammation. Conversely, inhibiting PP2A activity during smoke exposure exacerbated inflammatory responses in the lung. To further determine how PP2A modulates the responses to cigarette smoke in the lung, enzyme levels were manipulated in SAE cells using protein transfection and short hairpin RNA (shRNA) techniques. Increasing PP2A activity in SAE cells via PP2A protein transfection downregulated cytokine expression and prevented the induction of proteases following cigarette smoke extract (CSE) treatment. Conversely, decreasing enzymatic activity by stably transfecting SAE cells with shRNA for the A subunit of PP2A exacerbated these smoke-mediated responses. This study establishes that PP2A induction by cigarette smoke modulates immune and proteolytic responses to cigarette smoke exposure. Together, these findings suggest that manipulation of PP2A activity may be a plausible means to treat COPD and other inflammatory diseases.

Patient specific proteolytic activity of monocyte-derived macrophages and osteoclasts predicted with temporal kinase activation states during differentiation

Patient-to-patient variability in disease progression continues to complicate clinical decisions of treatment regimens for cardiovascular diseases, metastatic cancers and osteoporosis. Here, we investigated if monocytes, circulating white blood cells that enter tissues and contribute to disease progression by differentiating into macrophages or osteoclasts, could be useful in understanding this variability. Monocyte-derived macrophages and osteoclasts produce cysteine cathepsins, powerful extracellular matrix proteases which have been mechanistically linked to accelerated atherosclerotic, osteoporotic, and tumor progression. We hypothesized that multivariate analysis of temporal kinase activation states during monocyte differentiation could predict cathepsin proteolytic responses of monocyte-derived macrophages and osteoclasts in a patient-specific manner. Freshly isolated primary monocytes were differentiated with M-CSF or RANKL into macrophages or osteoclasts, respectively, and phosphorylation of ERK1/2, Akt, p38 MAPK, JNK, c-jun, and IκB-α were measured at days 1, 3, 6, and 9. In parallel, cell diameters and numbers of nuclei were measured, and multiplex cathepsin zymography was used to quantify cathepsins K, L, S, and V activity from cell extracts and conditioned media. There was extensive patient-to-patient variability in temporal kinase activation states, cell morphologies, and cathepsin K, L, S, and V proteolytic activity. Partial least squares regression models trained with temporal kinase activation states successfully predicted patient-specific morphological characteristics (mean cell diameter and number of nuclei) and patient-specific cathepsin proteolytic activity with predictability as high as 95%, even with the challenge of incorporating the complex, unknown cues from individual patients' unique genetic and biochemical backgrounds. This personalized medicine approach considers patient variability in kinase signals to predict cathepsin activity. Such analyses may provide beneficial tools for personalized kinase and protease inhibitor therapies for tissue destructive diseases.

FRET peptides reveal differential proteolytic activation in intraerythrocytic stages of the malaria parasites Plasmodium berghei and Plasmodium yoelii

Malaria is still a major health problem in developing countries. It is caused by the protist parasite Plasmodium, in which proteases are activated during the cell cycle. Ca2+ is a ubiquitous signalling ion that appears to regulate protease activity through changes in its intracellular concentration. Proteases are crucial to Plasmodium development, but the role of Ca2+ in their activity is not fully understood. Here we investigated the role of Ca2+ in protease modulation among rodent Plasmodium spp. Using fluorescence resonance energy transfer (FRET) peptides, we verified protease activity elicited by Ca2+ from the endoplasmatic reticulum (ER) after stimulation with thapsigargin (a sarco/endoplasmatic reticulum Ca2+-ATPase (SERCA) inhibitor) and from acidic compartments by stimulation with nigericin (a K+/H+ exchanger) or monensin (a Na+/H+ exchanger). Intracellular (BAPTA/AM) and extracellular (EGTA) Ca2+ chelators were used to investigate the role played by Ca2+ in protease activation. In Plasmodium berghei both EGTA and BAPTA blocked protease activation, whilst in Plasmodium yoelii these compounds caused protease activation. The effects of protease inhibitors on thapsigargin-induced proteolysis also differed between the species. Pepstatin A and phenylmethylsulphonyl fluoride (PMSF) increased thapsigargin-induced proteolysis in P. berghei but decreased it in P. yoelii. Conversely, E64 reduced proteolysis in P. berghei but stimulated it in P. yoelii. The data point out key differences in proteolytic responses to Ca2+ between species of Plasmodium.