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Sample records for caspase-3-dependent proteolytic activation

  1. Environmental neurotoxin dieldrin induces apoptosis via caspase-3-dependent proteolytic activation of protein kinase C delta (PKCdelta): Implications for neurodegeneration in Parkinson's disease

    PubMed Central

    Kanthasamy, Anumantha G; Kitazawa, Masashi; Yang, Yongjie; Anantharam, Vellareddy; Kanthasamy, Arthi

    2008-01-01

    Background In previous work, we investigated dieldrin cytotoxicity and signaling cell death mechanisms in dopaminergic PC12 cells. Dieldrin has been reported to be one of the environmental factors correlated with Parkinson's disease and may selectively destroy dopaminergic neurons. Methods Here we further investigated dieldrin toxicity in a dopaminergic neuronal cell model of Parkinson's disease, namely N27 cells, using biochemical, immunochemical, and flow cytometric analyses. Results In this study, dieldrin-treated N27 cells underwent a rapid and significant increase in reactive oxygen species followed by cytochrome c release into cytosol. The cytosolic cytochrome c activated caspase-dependent apoptotic pathway and the increased caspase-3 activity was observed following a 3 hr dieldrin exposure in a dose-dependent manner. Furthermore, dieldrin caused the caspase-dependent proteolytic cleavage of protein kinase C delta (PKCδ) into 41 kDa catalytic and 38 kDa regulatory subunits in N27 cells as well as in brain slices. PKCδ plays a critical role in executing the apoptotic process in dieldrin-treated dopaminergic neuronal cells because pretreatment with the PKCδ inhibitor rottlerin, or transfection and over-expression of catalytically inactive PKCδK376R, significantly attenuates dieldrin-induced DNA fragmentation and chromatin condensation. Conclusion Together, we conclude that caspase-3-dependent proteolytic activation of PKCδ is a critical event in dieldrin-induced apoptotic cell death in dopaminergic neuronal cells. PMID:18945348

  2. Environmental neurotoxin dieldrin induces apoptosis via caspase-3-dependent proteolytic activation of protein kinase C delta (PKCdelta): Implications for neurodegeneration in Parkinson's disease.

    PubMed

    Kanthasamy, Anumantha G; Kitazawa, Masashi; Yang, Yongjie; Anantharam, Vellareddy; Kanthasamy, Arthi

    2008-10-22

    In previous work, we investigated dieldrin cytotoxicity and signaling cell death mechanisms in dopaminergic PC12 cells. Dieldrin has been reported to be one of the environmental factors correlated with Parkinson's disease and may selectively destroy dopaminergic neurons. Here we further investigated dieldrin toxicity in a dopaminergic neuronal cell model of Parkinson's disease, namely N27 cells, using biochemical, immunochemical, and flow cytometric analyses. In this study, dieldrin-treated N27 cells underwent a rapid and significant increase in reactive oxygen species followed by cytochrome c release into cytosol. The cytosolic cytochrome c activated caspase-dependent apoptotic pathway and the increased caspase-3 activity was observed following a 3 hr dieldrin exposure in a dose-dependent manner. Furthermore, dieldrin caused the caspase-dependent proteolytic cleavage of protein kinase C delta (PKCδ) into 41 kDa catalytic and 38 kDa regulatory subunits in N27 cells as well as in brain slices. PKCδ plays a critical role in executing the apoptotic process in dieldrin-treated dopaminergic neuronal cells because pretreatment with the PKCδ inhibitor rottlerin, or transfection and over-expression of catalytically inactive PKCδ(K)³⁷⁶(R), significantly attenuates dieldrin-induced DNA fragmentation and chromatin condensation. Together, we conclude that caspase-3-dependent proteolytic activation of PKCδ is a critical event in dieldrin-induced apoptotic cell death in dopaminergic neuronal cells.

  3. Dieldrin induces apoptosis by promoting caspase-3-dependent proteolytic cleavage of protein kinase Cdelta in dopaminergic cells: relevance to oxidative stress and dopaminergic degeneration.

    PubMed

    Kitazawa, M; Anantharam, V; Kanthasamy, A G

    2003-01-01

    We previously reported that dieldrin, one of the potential environmental risk factors for development of Parkinson's disease, induces apoptosis in dopaminergic cells by generating oxidative stress. Here, we demonstrate that the caspase-3-dependent proteolytic activation of protein kinase Cdelta (PKCdelta) mediates as well as regulates the dieldrin-induced apoptotic cascade in dopaminergic cells. Exposure of PC12 cells to dieldrin (100-300 microM) results in the rapid release of cytochrome C, followed by the activation of caspase-9 and caspase-3 in a time- and dose-dependent manner. The superoxide dismutase mimetic Mn(III)tetrakis(4-benzoic acid)porphyrin chloride significantly attenuates dieldrin-induced cytochrome C release, indicating that reactive oxygen species may contribute to the activation of pro-apoptotic factors. Interestingly, dieldrin proteolytically cleaves native PKCdelta into a 41 kDa catalytic subunit and a 38 kDa regulatory subunit to activate the kinase. The dieldrin-induced proteolytic cleavage of PKCdelta and induction of kinase activity are completely inhibited by pretreatment with 50-100 microM concentrations of the caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK) and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (Z-DEVD-FMK), indicating that the proteolytic activation of PKCdelta is caspase-3-dependent. Additionally, Z-VAD-FMK, Z-DEVD-FMK or the PKCdelta specific inhibitor rottlerin almost completely block dieldrin-induced DNA fragmentation. Because dieldrin dramatically increases (40-80-fold) caspase-3 activity, we examined whether proteolytically activated PKCdelta amplifies caspase-3 via positive feedback activation. The PKCdelta inhibitor rottlerin (3-20 microM) dose-dependently attenuates dieldrin-induced caspase-3 activity, suggesting positive feedback activation of caspase-3 by PKCdelta. Indeed, delivery of catalytically active recombinant PKCdelta via a protein delivery system significantly

  4. Procaspase-activating compound 1 induces a caspase-3-dependent cell death in cerebellar granule neurons

    SciTech Connect

    Aziz, Gulzeb; Akselsen, Oyvind W.; Hansen, Trond V.; Paulsen, Ragnhild E.

    2010-09-15

    Procaspase-activating compound 1, PAC-1, has been introduced as a direct activator of procaspase-3 and has been suggested as a therapeutic agent against cancer. Its activation of procaspase-3 is dependent on the chelation of zinc. We have tested PAC-1 and an analogue of PAC-1 as zinc chelators in vitro as well as their ability to activate caspase-3 and induce cell death in chicken cerebellar granule neuron cultures. These neurons are non-dividing, primary cells with normal caspase-3. The results reported herein show that PAC-1 chelates zinc, activates procaspase-3, and leads to caspase-3-dependent cell death in neurons, as the specific caspase-3-inhibitor Ac-DEVD-cmk inhibited both the caspase-3 activity and cell death. Thus, chicken cerebellar granule neurons is a suitable model to study mechanisms of interference with apoptosis of PAC-1 and similar compounds. Furthermore, the present study also raises concern about potential neurotoxicity of PAC-1 if used in cancer therapy.

  5. Caspase 3 - Dependent Proteolytic Cleavage of Tau Causes Neurofibrillary Tangles and Results in Cognitive Impairment During Normal Aging

    PubMed Central

    Means, John C.; Gerdes, Bryan C.; Kaja, Simon; Sumien, Nathalie; Payne, Andrew J.; Stark, Danny A.; Borden, Priscilla K.; Price, Jeffrey L.; Koulen, Peter

    2016-01-01

    Mouse models of neurodegenerative diseases such as Alzheimer’s disease (AD) are important for understanding how pathological signaling cascades change neural circuitry and in time interrupt cognitive function. Here, we introduce a non-genetic preclinical model for aging and show that it exhibits cleaved tau protein, active caspases and neurofibrillary tangles, hallmarks of AD, causing behavioral deficits measuring cognitive impairment. To our knowledge this is the first report of a non-transgenic, non-interventional mouse model displaying structural, functional and molecular aging deficits associated with AD and other tauopathies in humans with potentially high impact on both new basic research into pathogenic mechanisms and new translational research efforts. Tau aggregation is a hallmark of tauopathies, including AD. Recent studies have indicated that cleavage of tau plays an important role in both tau aggregation and disease. In this study we use wild type mice as a model for normal aging and resulting age-related cognitive impairment. We provide evidence that aged mice have increased levels of activated caspases, which significantly correlates to increased levels of truncated tau and formation of neurofibrillary tangles. In addition, cognitive decline was significantly negatively associated with increased levels of caspase activity and tau truncated by caspase-3. Experimentally induced inhibition of caspases prevented this proteolytic cleavage of tau truncation and the associated formation of neurofibrillary tangles. Our study shows the strength of using a non-transgenic model to study structure, function and molecular mechanisms in aging and age related diseases of the brain. PMID:27220334

  6. LPS inhibits caspase 3-dependent apoptosis in RAW264.7 macrophages induced by the AMPK activator AICAR

    SciTech Connect

    Russe, Otto Quintus Möser, Christine V. Kynast, Katharina L. King, Tanya S. Olbrich, Katrin Grösch, Sabine Geisslinger, Gerd Niederberger, Ellen

    2014-05-09

    Highlights: • AMPK-activation induces caspase 3-dependent apoptosis in macrophages. • Apoptosis is associated with decreased mTOR and increased p21 levels. • All effects can be significantly inhibited by the TLR4 agonist lipopolysaccharide. - Abstract: AMP-activated kinase is a cellular energy sensor which is activated in stages of increased ATP consumption. Its activation has been associated with a number of beneficial effects such as decreasing inflammatory processes and the disease progress of diabetes and obesity, respectively. Furthermore, AMPK activation has been linked with induction of cell cycle arrest and apoptosis in cancer and vascular cells, indicating that it might have a therapeutic impact for the treatment of cancer and atherosclerosis. However, the impact of AMPK on the proliferation of macrophages, which also play a key role in the formation of atherosclerotic plaques and in inflammatory processes, has not been focused so far. We have assessed the influence of AICAR- and metformin-induced AMPK activation on cell viability of macrophages with and without inflammatory stimulation, respectively. In cells without inflammatory stimulation, we found a strong induction of caspase 3-dependent apoptosis associated with decreased mTOR levels and increased expression of p21. Interestingly, these effects could be inhibited by co-stimulation with bacterial lipopolysaccharide (LPS) but not by other proinflammatory cytokines suggesting that AICAR induces apoptosis via AMPK in a TLR4-pathway dependent manner. In conclusion, our results revealed that AMPK activation is not only associated with positive effects but might also contribute to risk factors by disturbing important features of macrophages. The fact that LPS is able to restore AMPK-associated apoptosis might indicate an important role of TLR4 agonists in preventing unfavorable cell death of immune cells.

  7. Ethanol inhibits methionine adenosyltransferase II activity and S-adenosylmethionine biosynthesis and enhances caspase-3-dependent cell death in T lymphocytes: relevance to alcohol-induced immunosuppression.

    PubMed

    Hote, Prachi T; Sahoo, Rashmita; Jani, Tanvi S; Ghare, Smita S; Chen, Theresa; Joshi-Barve, Swati; McClain, Craig J; Barve, Shirish S

    2008-06-01

    An important aspect in alcohol abuse-associated immune suppression is the loss of T helper CD4(+) lymphocytes, leading to impairment of multiple immune functions. Our work has shown that ethanol can sensitize CD4(+) T lymphocytes to caspase-3-dependent activation-induced cell death (AICD). It has been demonstrated that the formation of S-adenosylmethionine (SAMe) catalyzed by methionine adenosyltransferase (MAT) II is essential for CD4(+) T-cell activation and proliferation. Since ethanol is known to affect SAMe metabolism in hepatocytes, we investigated the effect of ethanol on MAT II activity/expression, SAMe biosynthesis and cell survival in CD4(+) T lymphocytes. We demonstrate for the first time that ethanol at a physiologically relevant concentration (25 mM) substantially decreased the enzymatic activity of MAT II in T lymphocytes. Ethanol was observed to decrease the transcription of MAT2A, which encodes the catalytic subunit of MAT II and is vital for MAT II activity and SAMe biosynthesis. Furthermore, correspondent to its effect on MAT II, ethanol decreased intracellular SAMe levels and enhanced caspase-3-dependent AICD. Importantly, restoration of intracellular SAMe levels by exogenous SAMe supplementation considerably decreased both caspase-3 activity and apoptotic death in T lymphocytes. In conclusion, our data show that MAT II and SAMe are critical molecular components essential for CD4(+) T-cell survival that are affected by ethanol, leading to enhanced AICD. Furthermore, these studies provide a clinical paradigm for the development of much needed therapy using SAMe supplementation in the treatment of immune dysfunction induced by alcohol abuse.

  8. Caspase-3 dependent nitrergic neuronal apoptosis following cavernous nerve injury is mediated via RhoA and ROCK activation in major pelvic ganglion.

    PubMed

    Hannan, Johanna L; Matsui, Hotaka; Sopko, Nikolai A; Liu, Xiaopu; Weyne, Emmanuel; Albersen, Maarten; Watson, Joseph W; Hoke, Ahmet; Burnett, Arthur L; Bivalacqua, Trinity J

    2016-07-08

    Axonal injury due to prostatectomy leads to Wallerian degeneration of the cavernous nerve (CN) and erectile dysfunction (ED). Return of potency is dependent on axonal regeneration and reinnervation of the penis. Following CN injury (CNI), RhoA and Rho-associated protein kinase (ROCK) increase in penile endothelial and smooth muscle cells. Previous studies indicate that nerve regeneration is hampered by activation of RhoA/ROCK pathway. We evaluated the role of RhoA/ROCK pathway in CN regulation following CNI using a validated rat model. CNI upregulated gene and protein expression of RhoA/ROCK and caspase-3 mediated apoptosis in the major pelvic ganglion (MPG). ROCK inhibitor (ROCK-I) prevented upregulation of RhoA/ROCK pathway as well as activation of caspase-3 in the MPG. Following CNI, there was decrease in the dimer to monomer ratio of neuronal nitric oxide synthase (nNOS) protein and lowered NOS activity in the MPG, which were prevented by ROCK-I. CNI lowered intracavernous pressure and impaired non-adrenergic non-cholinergic-mediated relaxation in the penis, consistent with ED. ROCK-I maintained the intracavernous pressure and non-adrenergic non-cholinergic-mediated relaxation in the penis following CNI. These results suggest that activation of RhoA/ROCK pathway mediates caspase-3 dependent apoptosis of nitrergic neurons in the MPG following CNI and that ROCK-I can prevent post-prostatectomy ED.

  9. Caspase-3 dependent nitrergic neuronal apoptosis following cavernous nerve injury is mediated via RhoA and ROCK activation in major pelvic ganglion

    PubMed Central

    Hannan, Johanna L.; Matsui, Hotaka; Sopko, Nikolai A.; Liu, Xiaopu; Weyne, Emmanuel; Albersen, Maarten; Watson, Joseph W.; Hoke, Ahmet; Burnett, Arthur L.; Bivalacqua, Trinity J.

    2016-01-01

    Axonal injury due to prostatectomy leads to Wallerian degeneration of the cavernous nerve (CN) and erectile dysfunction (ED). Return of potency is dependent on axonal regeneration and reinnervation of the penis. Following CN injury (CNI), RhoA and Rho-associated protein kinase (ROCK) increase in penile endothelial and smooth muscle cells. Previous studies indicate that nerve regeneration is hampered by activation of RhoA/ROCK pathway. We evaluated the role of RhoA/ROCK pathway in CN regulation following CNI using a validated rat model. CNI upregulated gene and protein expression of RhoA/ROCK and caspase-3 mediated apoptosis in the major pelvic ganglion (MPG). ROCK inhibitor (ROCK-I) prevented upregulation of RhoA/ROCK pathway as well as activation of caspase-3 in the MPG. Following CNI, there was decrease in the dimer to monomer ratio of neuronal nitric oxide synthase (nNOS) protein and lowered NOS activity in the MPG, which were prevented by ROCK-I. CNI lowered intracavernous pressure and impaired non-adrenergic non-cholinergic-mediated relaxation in the penis, consistent with ED. ROCK-I maintained the intracavernous pressure and non-adrenergic non-cholinergic-mediated relaxation in the penis following CNI. These results suggest that activation of RhoA/ROCK pathway mediates caspase-3 dependent apoptosis of nitrergic neurons in the MPG following CNI and that ROCK-I can prevent post-prostatectomy ED. PMID:27388816

  10. Increased anticancer activity of the thymidylate synthase inhibitor BGC9331 combined with the topoisomerase I inhibitor SN-38 in human colorectal and breast cancer cells: induction of apoptosis and ROCK cleavage through caspase-3-dependent and -independent mechanisms.

    PubMed

    Coudray, Anne-Marie; Louvet, Christophe; Kornprobst, Michel; Raymond, Eric; André, Thierry; Tournigand, Christophe; Faivre, Sandrine; De Gramont, Aimery; Larsen, Annette K; Gespach, Christian

    2005-08-01

    The folate analogue BGC9331 is a new thymidylate synthase (TS) inhibitor showing a broad spectrum of cyto-toxic activity against several human solid tumors, including colorectal cancer. In this study, we investigated the anticancer activity of BGC9331 either alone or combined with 5-fluorouracil (5-FU), MTA (multi-target antifolate), oxali-platin and SN-38, the active metabolite of the topoisomerase I inhibitor CPT-11. The antiproliferative activity of each drug and BGC9331-based combinations was investigated in the HT-29 human colorectal cancer cell line and its HT-29/5-FU counterparts selected for resistance to 5-FU. BGC9331 combined with MTA or SN-38 induced synergistic responses in HT-29 cells. Treatment of HT-29 cells with either BGC9331 or SN-38 increased caspase-3 activity and the percentage of apoptotic cells from 3 to 13%. Both drugs also augmented the proteolytic cleavage of the Rho-kinase ROCK-1 that was attenuated by the caspase-3 pathway inhibitor z-DEVD-fmk. BGC9331 combined with SN-38 further increased the percentage of apoptotic cells to 25%, and inhibited cell cycle progression and cell proliferation by 65%. This was accompanied by proteolytic activation of ROCK-1, through both caspase-3-dependent and -independent mechanisms, as shown in caspase-3-deficient MCF-7 breast cancer cells. These encouraging results warrant further preclinical investigations and clinical trials on the use of BGC9331 combined with SN-38/CPT-11 in treatment of patients with advanced colorectal or gastric cancers.

  11. ETHANOL INHIBITS METHIONINE ADENOSYLTRANSFERASE II (MAT II) ACTIVITY AND S-ADENOSYLMETHIONINE (SAMe) BIOSYNTHESIS AND ENHANCES CASPASE-3 DEPENDENT CELL DEATH IN T LYMHOCYTES: RELEVANCE TO ALCOHOL INDUCED IMMUNOSUPPRESSION

    PubMed Central

    Hote, Prachi T.; Sahoo, Rashmita; Jani, Tanvi S.; Ghare, Smita S.; Chen, Theresa; Joshi-Barve, Swati; McClain, Craig J.; Barve, Shirish S.

    2015-01-01

    An important aspect in alcohol abuse associated immune suppression is the loss of T helper CD4+ lymphocytes leading to an impairment of multiple immune functions. Our work has shown that ethanol can sensitize CD4+ T lymphocytes to activation-induced, caspase-3 dependent cell death (AICD). It has been demonstrated that formation of S-adenosylmethionine (SAMe) catalyzed by methionine adenosyltransferase II (MAT II) is essential for CD4+ T cell activation and proliferation. Since ethanol is known to affect SAMe metabolism in hepatocytes, we investigated the effect of ethanol on MAT II activity/expression, SAMe biosynthesis and cell survival in CD4+ T lymphocytes. We demonstrate for the first time that ethanol at a physiologically relevant concentration (25mM) substantially decreased the enzymatic activity of MAT II in T lymphocytes. Ethanol was observed to decrease the transcription of MAT2A, which encodes the catalytic subunit of MAT II and is vital for MAT II activity and SAMe biosynthesis. Further, correspondent to its effect on MAT II, ethanol decreased intracellular SAMe levels and enhanced caspase-3 dependent AICD. Importantly, restoration of intracellular SAMe levels by exogenous SAMe supplementation considerably decreased both caspase-3 activity and apoptotic death in T lymphocytes. In conclusion, our data shows that MAT II and SAMe are critical molecular components essential for CD4+ T cell survival which are affected by ethanol leading to enhanced AICD. Furthermore, these studies provide a clinical paradigm for the development of the much needed therapy using SAMe supplementation in the treatment of immune dysfunction induced by alcohol abuse. PMID:17869084

  12. UV-A Irradiation Activates Nrf2-Regulated Antioxidant Defense and Induces p53/Caspase3-Dependent Apoptosis in Corneal Endothelial Cells

    PubMed Central

    Liu, Cailing; Vojnovic, Dijana; Kochevar, Irene E.; Jurkunas, Ula V.

    2016-01-01

    Purpose To examine whether Nrf2-regulated antioxidant defense and p53 are activated in human corneal endothelial cells (CEnCs) by environmental levels of ultraviolet A (UV-A), a known stimulator of oxidative stress. Methods Immortalized human CEnCs (HCEnCi) were exposed to UV-A fluences of 2.5, 5, 10, or 25 J/cm2, then allowed to recover for 3 to 24 hours. Control HCEnCi did not receive UV-A. Reactive oxygen species (ROS) were measured using H2DCFDA. Cell cytotoxicity was evaluated by lactate dehydrogenase (LDH) release. Levels of Nrf2, HO-1, NQO-1, p53, and caspase3 were detected by immunnoblotting or real-time PCR. Activated caspase3 was measured by immunoblotting and a fluorescence assay. Results Exposure of HCEnCi to 5, 10, and 25 J/cm2 UV-A increased ROS levels compared with controls. Nrf2, HO-1, and NQO-1 mRNA increased 1.7- to 3.2-fold at 3 and 6 hours after irradiation with 2.5 and 5 J/cm2 UV-A. At 6 hours post irradiation, UV-A (5 J/cm2) enhanced nuclear Nrf2 translocation. At 24 hours post treatment, UV-A (5, 10, and 25 J/cm2) produced a 1.8- to 2.8-fold increase in phospho-p53 and a 2.6- to 6.0-fold increase in activated caspase3 compared with controls, resulting in 20% to 42% cell death. Conclusions Lower fluences of UV-A induce Nrf2-regulated antioxidant defense and higher fluences activate p53 and caspase3, indicating that even near-environmental levels of UV-A may affect normal CEnCs. This data suggest that UV-A may especially damage cells deficient in antioxidant defense, and thus may be involved in the etiology of Fuchs' endothelial corneal dystrophy (FECD). PMID:27127932

  13. Le Carbone, a charcoal supplement, modulates DSS-induced acute colitis in mice through activation of AMPKα and downregulation of STAT3 and caspase 3 dependent apoptotic pathways.

    PubMed

    Afrin, Mst Rejina; Arumugam, Somasundaram; Rahman, Md Azizur; Karuppagounder, Vengadeshprabhu; Sreedhar, Remya; Harima, Meilei; Suzuki, Hiroshi; Nakamura, Takashi; Miyashita, Shizuka; Suzuki, Kenji; Ueno, Kazuyuki; Watanabe, Kenichi

    2017-02-01

    Le Carbone (LC) is a charcoal supplement, which contains a large amount of dietary fibers. Several studies suggested that charcoal supplement may be beneficial for stomach disorders, diarrhea, gas and indigestion. But no studies address whether LC intake would suppress inflammation, cell proliferation or disease progression in colitis. In the present study, the effect of LC on experimental colitis induced by dextran sulfate sodium (DSS) in mice and its possible mechanism of action were examined. A study was designed for 8days, using C57BL/6 female mice that were administered with 3% DSS in drinking water for 7days followed by another 1day consumption of normal water with or without treatment. LC suspension was administered daily for 7days via oral gavage using 5mg/mouse in treatment group and normal group was supplied with drinking water. LC suspension significantly attenuated the loss of body weight and shortening of colon length induced by DSS. The disease activity index, histopathologic changes were significantly reduced by LC treatment. The inflammatory mediators TNFα, IL-1β, p-STAT3 and p-NF-κB induced in the colon by DSS were markedly suppressed by LC. The increased activation of AMPKα in the colon was also detected in LC group. Furthermore, the apoptotic marker protein cleaved caspase 3 was down-regulated and anti-apoptotic proteins Bcl2 and Bcl-xL were significantly up-regulated by LC treatment. Taken together, our results demonstrate the ability of LC to inhibit inflammation, apoptosis and give some evidence for its potential use as adjuvant treatment of inflammatory bowel disease.

  14. Dying glioma cells establish a proangiogenic microenvironment through a caspase 3 dependent mechanism.

    PubMed

    Feng, Xiao; Yu, Yang; He, Sijia; Cheng, Jin; Gong, Yanping; Zhang, Zhengxiang; Yang, Xuguang; Xu, Bing; Liu, Xinjian; Li, Chuan-Yuan; Tian, Ling; Huang, Qian

    2017-01-28

    Vascular recovery or re-angiogenesis after radiotherapy plays a significant role in tumor recurrence, whereas molecular mechanisms of this process remain elusive. In this work, we found that dying glioma cells promoted post-irradiation angiogenesis through a caspase 3 dependent mechanism. Evidence in vitro and in vivo indicated that caspase 3 inhibition undermined proangiogenic effects of dying glioma cells. Proteolytic inactivation of caspase 3 in glioma cells reduced tumorigenicity. Importantly, we identified that NF-κB/COX-2/PGE2 axis acted as downstream signaling of caspase 3, mediating proangiogenic response after irradiation. Additionally, VEGF-A, regulated by caspase 3 possibly through phosphorylated eIF4E, was recognized as another downstream factor participating in the proangiogenic response. In conclusion, these data demonstrated that caspase 3 in dying glioma cells supported the proangiogenic response after irradiation by governing NF-κB/COX-2/PGE2 axis and p-eIF4E/VEGF-A signaling. While inducing caspase 3 activation has been a generally-adopted notion in cancer therapeutics, our study counterintuitively illustrated that caspase 3 activation in dying glioma cells unfavorably supported post-irradiation angiogenesis. This double-edged role of caspase 3 suggested that taming caspase 3 from the opposite side, not always activating it, may provide novel therapeutic strategies due to restricted post-irradiation angiogenesis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  15. Positive feedback of protein kinase C proteolytic activation during apoptosis.

    PubMed Central

    Leverrier, Sabrina; Vallentin, Alice; Joubert, Dominique

    2002-01-01

    In contrast with protein kinase Calpha (PKCalpha) and PKCepsilon, which are better known for promoting cell survival, PKCdelta is known for its pro-apoptotic function, which is exerted mainly through a caspase-3-dependent proteolytic activation pathway. In the present study, we used the rat GH3B6 pituitary adenoma cell line to show that PKCalpha and PKCepsilon are activated and relocalized together with PKCdelta when apoptosis is induced by a genotoxic stress. Proteolytic activation is a crucial step used by the three isoforms since: (1) the catalytic domains of the PKCalpha, PKCepsilon or PKCdelta isoforms (CDalpha, CDepsilon and CDdelta respectively) accumulated, and this accumulation was dependent on the activity of both calpain and caspase; and (2) transient expression of CDalpha, CDepsilon or CDdelta sufficed to induce apoptosis. However, following this initial step of proteolytic activation, the pathways diverge; cytochrome c release and caspase-3 activation are induced by CDepsilon and CDdelta, but not by CDalpha. Another interesting finding of the present study is the proteolysis of PKCdelta induced by CDepsilon expression that revealed the existence of a cross-talk between PKC isoforms during apoptosis. Hence the PKC family may participate in the apoptotic process of pituitary adenoma cells at two levels: downstream of caspase and calpain, and via retro-activation of caspase-3, resulting in the amplification of its own proteolytic activation. PMID:12238950

  16. Proteolytic activities in yeast.

    PubMed

    Saheki, T; Holzer, H

    1975-03-28

    Studies on the mechanism and time course of the activation of proteinases A (EC 3.4.23.8), B (EC 3.4.22.9) and C (EC 3.4.12.--) in crude yeast extracts at pH 5.1 and 25 degrees C showed that the increase in proteinase B activity is paralleled with the disappearance of proteinase B inhibitor. Addition of purified proteinase A to fresh crude extracts accelerates the inactivation of the proteinase B inhibitor and the appearance of maximal activities of proteinases B and C. The decrease of proteinase B inhibitor activity and the increase of proteinase B activity are markedly retarded by the addition of pepstatin. Because 10-minus 7 M pepstatin completely inhibits proteinase A without affecting proteinase B activity, this is another indication for the role of proteinase A during the activation of proteinase B. Whereas extracts of yeast grown on minimal medium reached maximal activation of proteinases B and C after 20 h of incubation at pH 5.1 and 25 degrees C, extracts of yeast grown on complete medium had to be incubated for about 100 h. In the latter case, the addition of proteinas A results in maximal activation of proteinases B and C and disappearance of proteinase B inhibitor activity only after 10--20 h of incubation. With the optimal conditions, the maximal activities of proteinases A, B and C, as well as of the proteinase B inhibitor, were determined in crude extracts of yeast that had been grown batchwise for different lengths of time either on minimal or on complete medium. Upon incubation, all three proteinases were activated by several times their initial activity. This reflects the existence of proteolytically degradable inhibitors of the three proteinases and together with the above mentioned observations it demonstrates that the "activation" of yeast proteinases A, B and C upon incubation results from the proteolytic digestion of inhibitors rather than from activation of inactive zymogens by limited proteolysis.

  17. Dihydroartemisinin (DHA) induces caspase-3-dependent apoptosis in human lung adenocarcinoma ASTC-a-1 cells.

    PubMed

    Lu, Ying-Ying; Chen, Tong-Sheng; Qu, Jun-Le; Pan, Wen-Liang; Sun, Lei; Wei, Xun-Bin

    2009-02-02

    Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, isolated from the traditional Chinese herb Artemisia annua, is recommended as the first-line anti-malarial drug with low toxicity. DHA has been shown to possess promising anticancer activities and induce cancer cell death through apoptotic pathways, although the molecular mechanisms are not well understood. In this study, cell counting kit (CCK-8) assay was employed to evaluate the survival of DHA-treated ASTC-a-1 cells. The induction of apoptosis was detected by Hoechst 33258 and PI staining as well as flow cytometry analysis. Collapse of mitochondrial transmembrane potential (DeltaPsim) was measured by dynamic detection under a laser scanning confocal microscope and flow cytometry analysis using Rhodamine123. Caspase-3 activities measured with or without Z-VAD-fmk (a broad spectrum caspase inhibitor) pretreatment by FRET techniques, caspase-3 activity measurement, and western blotting analysis. Our results indicated that DHA induced apoptotic cell death in a dose- and time-dependent manner, which was accompanied by mitochondrial morphology changes, the loss of DeltaPsim and the activation of caspase-3. These results show for the first time that DHA can inhibit proliferation and induce apoptosis via caspase-3-dependent mitochondrial death pathway in ASTC-a-1 cells. Our work may provide evidence for further studies of DHA as a possible anticancer drug in the clinical treatment of lung adenocarcinoma.

  18. Blocking caspase-3-dependent pathway preserves hair cells from salicylate-induced apoptosis in the guinea pig cochlea.

    PubMed

    Feng, Hao; Yin, Shi-Hua; Tang, An-Zhou

    2011-07-01

    In the present study, we aim to explore whether the caspase-3-dependent pathway is involved in the apoptotic cell death that occurs in the hair cells (HCs) of guinea pig cochlea following a salicylate treatment. Guinea pigs received sodium salicylate (Na-SA), at a dose of 200 mg·kg(-1)·d(-1) i.p., as a vehicle for 5 consecutive days. In some experiments, N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (zDEVD-FMK), a specific apoptosis inhibitor, was directly applied into the cochlea via the round window niche (RWN) prior to salicylate treatment for determination of caspase-3 activation. Alterations in auditory function were evaluated with auditory brainstem responses (ABR) thresholds. Caspase-3 activity was determined by measuring the proteolytic cleavage product of caspase-3 (N-terminated peptide substrate). DNA fragmentation within the nuclei was examined with a terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method. Ultrastructure variation in the target cell was assessed by electron microscopy (EM). Salicylate treatment initiated an obvious elevation in ABR thresholds with a maximum average shift of 60 dB sound pressure level (SPL), and caused significant apoptosis in both inner (IHCs) and outer (OHCs) hair cells resulted from an evident increasing in immunoreactivity to caspase-3 protease. Transmission electron microscopy (TEM) displayed chromatin condensation and nucleus margination accompanied by cell body shrinkage in the OHCs, but not in the IHCs. Scanning electron microscopy (SEM) showed breakdown, fusion, and loss in the stereociliary bundles at the apex of OHCs rather than IHCs. zDEVD-FMK pretreatment prior to salicylate injection substantially attenuated an expression of the apoptotic protease and protected HCs against apoptotic death, followed by a moderate relief in the thresholds of ABR, an alleviation in the submicroscopic structure was also identified. In particular, disorientation and insertion in the

  19. Proteolytic Activity in the Genus Ficus 1

    PubMed Central

    Williams, Donald C.; Sgarbieri, Valdemiro C.; Whitaker, John R.

    1968-01-01

    The latices of only 13 of a total of 46 species of Ficus examined contained appreciable proteolytic activity. Therefore, high proteolytic activity in the latex is not a distinguishing feature of the genus. The latex of F. stenocarpa had the highest specific activity followed closely by the latices of F. carica and F. glabrata. Latices of 6 species of Ficus were examined by chromatography on CM-cellulose and compared with the results obtained for 9 varieties of F. carica. All of the latices were found to contain multiple proteolytic enzymes. Chromatographically, the multiple enzyme components of the several varieties of F. carica were more similar than those of the several species examined. The latices of 16 varieties of F. carica were all different as determined by free boundary electrophoresis although the specific proteolytic activity of the latices was reasonably constant. PMID:16656886

  20. Aggregatibacter actinomycetemcomitans Infection Enhances Apoptosis In Vivo through a Caspase-3-Dependent Mechanism in Experimental Periodontitis

    PubMed Central

    Kang, Jun; de Brito Bezerra, Beatriz; Pacios, Sandra; Andriankaja, Oelisoa; Li, Yu; Tsiagbe, Vincent; Schreiner, Helen; Fine, Daniel H.

    2012-01-01

    The purpose of this study was to test the hypothesis that diabetes aggravates periodontal destruction induced by Aggregatibacter actinomycetemcomitans infection. Thirty-eight diabetic and 33 normal rats were inoculated with A. actinomycetemcomitans and euthanized at baseline and at 4, 5, and 6 weeks after inoculation. Bone loss and the infiltration of polymorphonuclear leukocytes (PMNs) in gingival epithelium were measured in hematoxylin-eosin-stained sections. The induction of tumor necrosis factor alpha (TNF-α) was evaluated by immunohistochemistry and of apoptotic cells by a TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay. After A. actinomycetemcomitans infection, the bone loss in diabetic rats was 1.7-fold and the PMN infiltration 1.6-fold higher than in normoglycemic rats (P < 0.05). The induction of TNF-α was 1.5-fold higher and of apoptotic cells was up to 3-fold higher in diabetic versus normoglycemic rats (P < 0.05). Treatment with a caspase-3 inhibitor significantly blocked noninflammatory cell apoptosis induced by A. actinomycetemcomitans infection in gingival epithelium and connective tissue (P < 0.05). These results provide new insight into how diabetes aggravates A. actinomycetemcomitans-induced periodontal destruction in rats by significantly increasing the inflammatory response, leading to increased bone loss and enhancing apoptosis of gingival epithelial and connective tissue cells through a caspase-3-dependent mechanism. Antibiotics had a more pronounced effect on many of these parameters in diabetic than in normoglycemic rats, suggesting a deficiency in the capacity of diabetic animals to resist infection. PMID:22451521

  1. Plasma proteolytic activity in liver transplant rejection.

    PubMed

    Scholz, T; Gallimore, M J; Bäckman, L; Mathisen, O; Bergan, A; Klintmalm, G B; Aasen, A O

    1999-01-01

    In this study, we evaluated the role of proteolytic enzymes belonging to the coagulation, fibrinolytic, and plasma contact systems in the early postoperative phase after orthotopic liver transplantation (OLT). Twenty-nine patients were studied at the time of OLT and during the first 2 postoperative weeks. Blood samples were collected daily after OLT and analyzed for kallikrein-like activity (KK), functional kallikrein inhibition (KKI), plasmin-like activity (PL), and alpha2-antiplasmin (AP). In addition, prekallikrein (PKK), prothrombin (PTH), antithrombin III (AT III), plasminogen (PLG), prothrombin/antithrombin III complexes (TAT), prothrombin fragment 1 + 2 (F1 + 2), and plasmin/alpha2-antiplasmin complexes (PAP) were measured. Nineteen patients experienced biopsy-verified acute rejections (AR) and ten patients had uneventful courses and served as controls. Plasma analyses showed that the contact, coagulation, and fibrinolytic systems were activated during OLT. Following OLT, continuous thrombin and plasmin generation was observed, and these effects were more pronounced in the group having an uneventful course than in patients with AR. Factors that could possibly affect plasma proteolytic activity, such as blood product usage during and after OLT and cold ischemia time of the liver graft, did not differ between the groups, nor did the routine liver function tests, alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

  2. Proteolytic Processing Regulates Placental Growth Factor Activities*

    PubMed Central

    Hoffmann, Daniel C.; Willenborg, Sebastian; Koch, Manuel; Zwolanek, Daniela; Müller, Stefan; Becker, Ann-Kathrin A.; Metzger, Stephanie; Ehrbar, Martin; Kurschat, Peter; Hellmich, Martin; Hubbell, Jeffrey A.; Eming, Sabine A.

    2013-01-01

    Placental growth factor (PlGF) is a critical mediator of blood vessel formation, yet mechanisms of its action and regulation are incompletely understood. Here we demonstrate that proteolytic processing regulates the biological activity of PlGF. Specifically, we show that plasmin processing of PlGF-2 yields a protease-resistant core fragment comprising the vascular endothelial growth factor receptor-1 binding site but lacking the carboxyl-terminal domain encoding the heparin-binding domain and an 8-amino acid peptide encoded by exon 7. We have identified plasmin cleavage sites, generated a truncated PlGF118 isoform mimicking plasmin-processed PlGF, and explored its biological function in comparison with that of PlGF-1 and -2. The angiogenic responses induced by the diverse PlGF forms were distinct. Whereas PlGF-2 increased endothelial cell chemotaxis, vascular sprouting, and granulation tissue formation upon skin injury, these activities were abrogated following plasmin digestion. Investigation of PlGF/Neuropilin-1 binding and function suggests a critical role for heparin-binding domain/Neuropilin-1 interaction and its regulation by plasmin processing. Collectively, here we provide new mechanistic insights into the regulation of PlGF-2/Neuropilin-1-mediated tissue vascularization and growth. PMID:23645683

  3. Nitric oxide-mediated apoptosis of neutrophils through caspase-8 and caspase-3-dependent mechanism

    PubMed Central

    Dubey, Megha; Nagarkoti, Sheela; Awasthi, Deepika; Singh, Abhishek K; Chandra, Tulika; Kumaravelu, J; Barthwal, Manoj K; Dikshit, Madhu

    2016-01-01

    Neutrophils play an indispensable role in killing of invading pathogens by enhancing reactive oxygen species (ROS) and NO generation, and subsequently undergoing apoptosis. Unlike ROS/NOX2, role of NO/NOS still remains undefined in the apoptosis of neutrophils (PMNs) and the present study attempts to decipher the importance of NO/NOS in the neutrophil apoptosis. Prolonged treatment of human PMNs or mice bone marrow derived neutrophils (BMDN) with NO led to enhanced ROS generation, caspase-8/caspase-3 cleavage, reduced mitochondrial membrane potential and finally cellular apoptosis. NO-induced ROS generation led to caspase-8 deglutathionylation and activation, which subsequently activated mitochondrial death pathway via BID (Bcl-2 family protein) cleavage. NO-mediated augmentation of caspase-8 and BID cleavage was significantly prevented in BMDN from neutrophil cytosolic factor-1 (NCF-1) knockout (KO) mice, implying the involvement of NOX2 in NO-induced apoptosis of PMNs. Furthermore, ROS, NO generation and inducible nitric oxide synthase (iNOS) expression were enhanced in a time-dependent manner in human PMNs and mice BMDN undergoing spontaneous apoptosis. Pharmacological and genetic ablation of iNOS in human PMNs and mice BMDN significantly reduced the levels of apoptosis. Impaired apoptosis of BMDN from iNOS KO mice was due to reduced caspase-8 activity which subsequently prevented caspase-3 and -9 activation. Altogether, our results suggest a crucial role of NO/iNOS in neutrophil apoptosis via enhanced ROS generation and caspase-8 mediated activation of mitochondrial death pathway. PMID:27584786

  4. Evaluation of proteolytic activity to differentiate some dematiaceous fungi.

    PubMed Central

    Espinel-Ingroff, A; Goldson, P R; McGinnis, M R; Kerkering, T M

    1988-01-01

    A total of 123 isolates of Cladosporium spp., Exophiala spp., Fonsecaea spp., Lecythophora hoffmannii, Phaeoannellomyces werneckii, Phialophora spp., Wangiella dermatitidis, and Xylohypha bantiana were tested for proteolytic activity by using 26 different formulations of gelatin, milk, casein, and Loeffler media. Other physiological properties examined included hydrolysis of tyrosine and xanthine, sodium nitrate utilization in Czapek Dox agar, and thermotolerance. Isolates of Exophiala jeanselmei, Fonsecaea compacta, Fonsecaea pedrosoi, W. dermatitidis, and X. bantiana lacked proteolytic activity. Proteolytic activity was variable among the remaining species, depending on the type of medium used. Thermotolerance had value in distinguishing some taxa. PMID:3343325

  5. Proteolytic activity in some Patagonian plants from Argentina.

    PubMed

    Sequeiros, Cynthia; López, Laura M I; Caffini, Néstor O; Natalucci, Claudia L

    2003-09-01

    Six Patagonian plants were screened for proteolytic activity: Colliguaja integerrima, Euphorbia collina, E. peplus and Stillingia patagonica (Euphorbiaceae), Philibertia gilliesii (Asclepiadaceae) and Grindelia chiloensis (Asteraceae). P. gilliesii extracts showed the highest specific activity, followed by S. patagonica and E. collina. Proteolytic activity was unnoticeable in the other three species studied. Inhibition assays revealed that P. gilliesii and S. patagonica extracts contain cysteine-type peptidases and that in E. collina serine-type peptidases are present.

  6. Proteolytic activity during senescence of plants.

    PubMed

    Huffaker, R C

    1990-01-01

    Although information has rapidly developed concerning the intracellular localization of plant proteins, relatively few reports concern the intracellular location of endo- and exo-proteolytic activities. Relatively few proteases have been purified, characterized, and associated with a specific cellular location. With the exception of the processing proteases involved in transport of proteins across membranes, little progress has yet been made concerning determination of in vivo products of specific proteases. Information on the turnover of individual proteins and the assessment of rate-limiting steps in pathways as proteins are turned over is steadily appearing. Since chloroplasts are the major site of both protein synthesis and, during senescence, degradation, it was important to show unambiguously that chloroplasts can degrade their own constituents. Another important contribution was to obtain evidence that the chloroplasts contain proteases capable of degrading their constituents. This work has been more tenuous because of the low activities found and the possibility of contamination by vacuolar enzymes during the isolation of organelles. The possible targeting of cytoplasmic proteins for degradation by facilitating their transport into vacuoles is a field which hopefully will develop more rapidly in the future. Information on targeting of proteins for degradation via the ubiquitin (Ub) degradation pathway is developing rapidly. Future research must determine how much unity exists across the different eukaryotic systems. At present, it has important implications for protein turnover in plants, since apparently Ub is involved in the degradation of phytochrome. Little information has been developed regarding what triggers increased proteolysis with the onset of senescence, although it appears to involve protein synthesis. Thus far, the evidence indicates that the complement of proteases prior to senescence is sufficient to carry out the observed protein

  7. Proteolytic activity during senescence of plants

    NASA Technical Reports Server (NTRS)

    Huffaker, R. C.

    1990-01-01

    Although information has rapidly developed concerning the intracellular localization of plant proteins, relatively few reports concern the intracellular location of endo- and exo-proteolytic activities. Relatively few proteases have been purified, characterized, and associated with a specific cellular location. With the exception of the processing proteases involved in transport of proteins across membranes, little progress has yet been made concerning determination of in vivo products of specific proteases. Information on the turnover of individual proteins and the assessment of rate-limiting steps in pathways as proteins are turned over is steadily appearing. Since chloroplasts are the major site of both protein synthesis and, during senescence, degradation, it was important to show unambiguously that chloroplasts can degrade their own constituents. Another important contribution was to obtain evidence that the chloroplasts contain proteases capable of degrading their constituents. This work has been more tenuous because of the low activities found and the possibility of contamination by vacuolar enzymes during the isolation of organelles. The possible targeting of cytoplasmic proteins for degradation by facilitating their transport into vacuoles is a field which hopefully will develop more rapidly in the future. Information on targeting of proteins for degradation via the ubiquitin (Ub) degradation pathway is developing rapidly. Future research must determine how much unity exists across the different eukaryotic systems. At present, it has important implications for protein turnover in plants, since apparently Ub is involved in the degradation of phytochrome. Little information has been developed regarding what triggers increased proteolysis with the onset of senescence, although it appears to involve protein synthesis. Thus far, the evidence indicates that the complement of proteases prior to senescence is sufficient to carry out the observed protein

  8. Proteolytic activity during senescence of plants

    NASA Technical Reports Server (NTRS)

    Huffaker, R. C.

    1990-01-01

    Although information has rapidly developed concerning the intracellular localization of plant proteins, relatively few reports concern the intracellular location of endo- and exo-proteolytic activities. Relatively few proteases have been purified, characterized, and associated with a specific cellular location. With the exception of the processing proteases involved in transport of proteins across membranes, little progress has yet been made concerning determination of in vivo products of specific proteases. Information on the turnover of individual proteins and the assessment of rate-limiting steps in pathways as proteins are turned over is steadily appearing. Since chloroplasts are the major site of both protein synthesis and, during senescence, degradation, it was important to show unambiguously that chloroplasts can degrade their own constituents. Another important contribution was to obtain evidence that the chloroplasts contain proteases capable of degrading their constituents. This work has been more tenuous because of the low activities found and the possibility of contamination by vacuolar enzymes during the isolation of organelles. The possible targeting of cytoplasmic proteins for degradation by facilitating their transport into vacuoles is a field which hopefully will develop more rapidly in the future. Information on targeting of proteins for degradation via the ubiquitin (Ub) degradation pathway is developing rapidly. Future research must determine how much unity exists across the different eukaryotic systems. At present, it has important implications for protein turnover in plants, since apparently Ub is involved in the degradation of phytochrome. Little information has been developed regarding what triggers increased proteolysis with the onset of senescence, although it appears to involve protein synthesis. Thus far, the evidence indicates that the complement of proteases prior to senescence is sufficient to carry out the observed protein

  9. FANCJ protein is important for the stability of FANCD2/FANCI proteins and protects them from proteasome and caspase-3 dependent degradation.

    PubMed

    Clark, David W; Tripathi, Kaushlendra; Dorsman, Josephine C; Palle, Komaraiah

    2015-10-06

    Fanconi anemia (FA) is a rare genome instability syndrome with progressive bone marrow failure and cancer susceptibility. FANCJ is one of 17 genes mutated in FA-patients, comprises a DNA helicase that is vital for properly maintaining genomic stability and is known to function in the FA-BRCA DNA repair pathway. While exact role(s) of FANCJ in this repair process is yet to be determined, it is known to interact with primary effector FANCD2. However, FANCJ is not required for FANCD2 activation but is important for its ability to fully respond to DNA damage. In this report, we determined that transient depletion of FANCJ adversely affects stability of FANCD2 and its co-regulator FANCI in multiple cell lines. Loss of FANCJ does not significantly alter cell cycle progression or FANCD2 transcription. However, in the absence of FANCJ, the majority of FANCD2 is degraded by both the proteasome and Caspase-3 dependent mechanism. FANCJ is capable of complexing with and stabilizing FANCD2 even in the absence of a functional helicase domain. Furthermore, our data demonstrate that FANCJ is important for FANCD2 stability and proper activation of DNA damage responses to replication blocks induced by hydroxyurea.

  10. FANCJ protein is important for the stability of FANCD2/FANCI proteins and protects them from proteasome and caspase-3 dependent degradation

    PubMed Central

    Clark, David W.; Tripathi, Kaushlendra; Dorsman, Josephine C.; Palle, Komaraiah

    2015-01-01

    Fanconi anemia (FA) is a rare genome instability syndrome with progressive bone marrow failure and cancer susceptibility. FANCJ is one of 17 genes mutated in FA-patients, comprises a DNA helicase that is vital for properly maintaining genomic stability and is known to function in the FA-BRCA DNA repair pathway. While exact role(s) of FANCJ in this repair process is yet to be determined, it is known to interact with primary effector FANCD2. However, FANCJ is not required for FANCD2 activation but is important for its ability to fully respond to DNA damage. In this report, we determined that transient depletion of FANCJ adversely affects stability of FANCD2 and its co-regulator FANCI in multiple cell lines. Loss of FANCJ does not significantly alter cell cycle progression or FANCD2 transcription. However, in the absence of FANCJ, the majority of FANCD2 is degraded by both the proteasome and Caspase-3 dependent mechanism. FANCJ is capable of complexing with and stabilizing FANCD2 even in the absence of a functional helicase domain. Furthermore, our data demonstrate that FANCJ is important for FANCD2 stability and proper activation of DNA damage responses to replication blocks induced by hydroxyurea. PMID:26336824

  11. Exposure to 1950-MHz TD-SCDMA Electromagnetic Fields Affects the Apoptosis of Astrocytes via Caspase-3-Dependent Pathway

    PubMed Central

    Li, Guo-qing; Zhang, Zhi-wen; Xue, Jing-hui; Liu, Hong-sheng; Zhu, Heng; Cheng, Ji-de; Liu, Yuan-ling; Li, An-ming; Zhang, Yi

    2012-01-01

    The usage of mobile phone increases globally. However, there is still a paucity of data about the impact of electromagnetic fields (EMF) on human health. This study investigated whether EMF radiation would alter the biology of glial cells and act as a tumor-promoting agent. We exposed rat astrocytes and C6 glioma cells to 1950-MHz TD-SCDMA for 12, 24 and 48 h respectively, and found that EMF exposure had differential effects on rat astroctyes and C6 glioma cells. A 48 h of exposure damaged the mitochondria and induced significant apoptosis of astrocytes. Moreover, caspase-3, a hallmark of apoptosis, was highlighted in astrocytes after 48 h of EMF exposure, accompanied by a significantly increased expression of bax and reduced level of bcl-2. The tumorigenicity assays demonstrated that astrocytes did not form tumors in both control and exposure groups. In contrast, the unexposed and exposed C6 glioma cells show no significant differences in both biological feature and tumor formation ability. Therefore, our results implied that exposure to the EMF of 1950-MHz TD-SCDMA may not promote the tumor formation, but continuous exposure damaged the mitochondria of astrocytes and induce apoptosis through a caspase-3-dependent pathway with the involvement of bax and bcl-2. PMID:22870319

  12. Quercetin-induced apoptosis acts through mitochondrial- and caspase-3-dependent pathways in human breast cancer MDA-MB-231 cells.

    PubMed

    Chien, Su-Yu; Wu, Yao-Chung; Chung, Jing-Gung; Yang, Jai-Sing; Lu, Hsu-Feng; Tsou, Mei-Fen; Wood, W G; Kuo, Shou-Jen; Chen, Dar-Ren

    2009-08-01

    There has been considerable evidence recently demonstrating the anti-tumour effects of flavonols. Quercetin, an ubiquitous bioactive flavonol, inhibits cells proliferation, induces cell cycle arrest and apoptosis in different cancer cell types. The precise molecular mechanism of quercetin-induced apoptosis in human breast cancer cells is unclear. The purpose of this study was to investigate effects of quercetin on cell viability and to determine its underlying mechanism in human breast cancer MDA-MB-231 cells. Quercetin decreased the percentage of viable cells in a dose- and time-dependent manner, which was associated with cell cycle arrest and apoptosis. Quercetin did not increase reactive oxygen species generation but increased cytosolic Ca(2+) levels and reduced the mitochondrial membrane potential (DeltaPsi(m)). Quercetin treatment promoted activation of caspase-3, -8 and -9 in MDA-MB-231 cells. Caspase inhibitors prevented the quercetin-induced loss of cell viability. Quercetin increased abundance of the pro-apoptotic protein Bax and decreased the levels of anti-apoptotic protein Bcl-2. Confocal laser microscope examination indicated that quercetin promoted apoptosis-inducing factor (AIF) release from mitochondria and stimulated translocation to the nucleus. Taken together, these findings suggest that quercetin results in human breast cancer MDA-MB-231 cell death through mitochondrial- and caspase-3-dependent pathways.

  13. Reinvestigation of the proteolytically active components of Bromelia pinguin fruit.

    PubMed

    Payrol, Juan Abreu; Obregón, Walter D; Natalucci, Claudia L; Caffini, Néstor O

    2005-09-01

    Pinguinain is the name given to a proteolytic enzyme preparation obtained from Bromelia pinguin fruits that has been scarcely studied. The present paper deals on the reexamination of the proteases present in fruits of B. pinguin grown in Cienfuegos, Cuba. The preparation (partially purified pinguinain, PPP) showed the main characteristics of the cysteine proteases, i.e., optimum pH within alkaline range (pH 7.2-8.8), inhibition of proteolytic activity by thiol blocking reagents, which is usually reverted by addition of cysteine, a remarkable thermal stability and notable stability at high ionic strength values. Isoelectric focusing and zymogram of PPP revealed the presence of several proteolytic components between pI 4.6 and 8.1. Preliminary peptidase purification by cationic exchange chromatography showed the presence of two main proteolytic fractions with molecular masses of approximately 20.0 kDa, according to SDS-PAGE.

  14. Gelatinase expression and proteolytic activity in giant‐cell arteritis

    PubMed Central

    Segarra, Marta; García‐Martínez, Ana; Sánchez, Montse; Hernández‐Rodríguez, José; Lozano, Ester; Grau, Josep M; Cid, Maria C

    2007-01-01

    Objectives Gelatinases (MMP2 and MMP9) are expressed in giant‐cell arteritis (GCA) and are thought to play a role in vessel disruption. However, their activation status and enzymatic activity have not been evaluated. Our aim was to investigate the distribution and proteolytic activity of gelatinases in GCA lesions at different stages. Methods Expression of MMP2, MMP9, MMP2‐activator MMP14 and their natural inhibitors TIMP1 and TIMP2 was determined by real‐time PCR and immunohistochemistry in temporal artery sections from 46 patients and 12 controls. MMP activation status and enzymatic activity were assessed by gelatin and film in situ zymography. Results Vascular smooth muscle cells from normal specimens constitutively expressed pro‐MMP2 and its inhibitor TIMP2 with no resulting proteolytic activity. In GCA MMP2, MMP9 and MMP14 were strongly expressed in their active form by infiltrating leucocytes. Inflamed arteries also expressed TIMP1 and TIMP2. However, the MMP9/TIMP1 and MMP2/TIMP2 ratios were higher in patients compared with controls, indicating an increased proteolytic balance in GCA which was confirmed by in situ zymography. Maximal gelatinase expression and activity occurred at the granulomatous areas surrounding the internal elastic lamina (IEL). Myointimal cells also expressed MMPs and exhibited proteolytic activity, suggesting a role for gelatinases in vascular remodelling and repair. Conclusions GCA lesions show intense expression of gelatinases. Activators and inhibitors are regulated to yield enhanced gelatinase activation and proteolytic activity. Distribution of expression and proteolytic activity suggests that gelatinases have a major role not only in the progression of inflammatory infiltrates and vessel destruction but also in vessel repair. PMID:17502363

  15. Assay for Lipolytic and Proteolytic Activity Using Marine Substrates

    PubMed Central

    Tom, Raymond A.; Crisan, Eli V.

    1975-01-01

    Nondestructive assay procedures for determining microbial lipolytic and proteolytic activity on marine substrates were developed and tested with 287 isolates of bacteria, filamentous fungi, and yeasts. A definite substrate specificity was noted when the enzymatic activities on marine and nonmarine substrates was compared. Of 170 lipolytic isolates, 14 were only active on menhaden oil, 11 could hydrolyze menhaden oil and Tween 80 and/or tributyrin, and 145 isolates could only hydrolyze one or both of the nonmarine lipids. Of the 198 proteolytic isolates, 10 were specific for codfish extract, 152 were active against the marine substrate plus casein and/or gelatin, and 36 were specific for nonmarine substrates. PMID:1167775

  16. Proteolytic activity of probiotic strain Lactobacillus helveticus M92.

    PubMed

    Beganović, Jasna; Kos, Blaženka; Leboš Pavunc, Andreja; Uroić, Ksenija; Džidara, Petra; Šušković, Jagoda

    2013-04-01

    The aim of this research was to investigate the potential of previously defined probiotic strain Lactobacillus helveticus M92 as functional starter culture for fermented dairy products. Therefore, proteolytic activity of L. helveticus M92 was investigated and compared with those of different representatives of probiotic and starter culture strains. Cluster analysis of AFLP fingerprints showed a difference of L. helveticus M92 compared to five other L. helveticus strains, but the percentage of similarity confirmed the identification on species level. Casein hydrolysis by L. helveticus M92 was monitored by agar-well diffusion test, SDS-PAGE and Anson's method. L. helveticus M92 exhibited the highest proteolytic activity among tested probiotic and starter cultures strains with the fastest acidification rate and the highest pH decrease after overnight incubation in skim milk. The presence of prtH2 gene was confirmed by PCR amplification with specific primers, while PCR product was not obtained after amplification with primers specific to prtH. Furthermore, SDS-PAGE LC-MS/MS analysis of insoluble proteome of L. helveticus M92 enabled identification of several proteins involved in proteolytic system of L. helveticus such as protease PrtM as well as proteins involved in Opp peptide transport system and the intracellular peptidases PepE, PepN, and PepQ.

  17. Epstein-Barr Virus MicroRNA miR-BART20-5p Suppresses Lytic Induction by Inhibiting BAD-Mediated caspase-3-Dependent Apoptosis.

    PubMed

    Kim, Hyoji; Choi, Hoyun; Lee, Suk Kyeong

    2015-11-18

    Epstein-Barr virus (EBV) is a human gammaherpesvirus associated with a variety of tumor types. EBV can establish latency or undergo lytic replication in host cells. In general, EBV remains latent in tumors and expresses a limited repertoire of latent proteins to avoid host immune surveillance. When the lytic cycle is triggered by some as-yet-unknown form of stimulation, lytic gene expression and progeny virus production commence. Thus far, the exact mechanism of EBV latency maintenance and the in vivo triggering signal for lytic induction have yet to be elucidated. Previously, we have shown that the EBV microRNA miR-BART20-5p directly targets the immediate early genes BRLF1 and BZLF1 as well as Bcl-2-associated death promoter (BAD) in EBV-associated gastric carcinoma. In this study, we found that both mRNA and protein levels of BRLF1 and BZLF1 were suppressed in cells following BAD knockdown and increased after BAD overexpression. Progeny virus production was also downregulated by specific knockdown of BAD. Our results demonstrated that caspase-3-dependent apoptosis is a prerequisite for BAD-mediated EBV lytic cycle induction. Therefore, our data suggest that miR-BART20-5p plays an important role in latency maintenance and tumor persistence of EBV-associated gastric carcinoma by inhibiting BAD-mediated caspase-3-dependent apoptosis, which would trigger immediate early gene expression. EBV has an ability to remain latent in host cells, including EBV-associated tumor cells hiding from immune surveillance. However, the exact molecular mechanisms of EBV latency maintenance remain poorly understood. Here, we demonstrated that miR-BART20-5p inhibited the expression of EBV immediate early genes indirectly, by suppressing BAD-induced caspase-3-dependent apoptosis, in addition to directly, as we previously reported. Our study suggests that EBV-associated tumor cells might endure apoptotic stress to some extent and remain latent with the aid of miR-BART20-5p. Blocking the

  18. In vivo Overhauser-enhanced MRI of proteolytic activity.

    PubMed

    Koonjoo, Neha; Parzy, Elodie; Massot, Philippe; Lepetit-Coiffé, Matthieu; Marque, Sylvain R A; Franconi, Jean-Michel; Thiaudiere, Eric; Mellet, Philippe

    2014-01-01

    There is an increasing interest in developing novel imaging strategies for sensing proteolytic activities in intact organisms in vivo. Overhauser-enhanced MRI (OMRI) offers the possibility to reveal the proteolysis of nitroxide-labeled macromolecules thanks to a sharp decrease of the rotational correlation time of the nitroxide moiety upon cleavage. In this paper, this concept is illustrated in vivo at 0.2 T using nitroxide-labeled elastin orally administered in mice. In vitro, this elastin derivative was OMRI-visible and gave rise to high Overhauser enhancements (19-fold at 18 mm nitroxide) upon proteolysis by pancreatic porcine elastase. In vivo three-dimensional OMRI detection of proteolysis was carried out. A keyhole fully balanced steady-state free precession sequence was used, which allowed 3D OMRI acquisition within 20 s at 0.125 mm(3) resolution. About 30 min after mouse gavage, proteolysis was detected in the duodenum, where Overhauser enhancements were 7.2 ± 2.4 (n = 7) and was not observed in the stomach. Conversely, orally administered free nitroxides or pre-digested nitroxide-labeled elastin were detected in the mouse's stomach by OMRI. Combined with specific molecular probes, this Overhauser-enhanced MRI technique can be used to evaluate unregulated proteolytic activities in various models of experimental diseases and for drug testing. Copyright © 2014 John Wiley & Sons, Ltd.

  19. Electrochemical Proteolytic Beacon for Detection of Matrix Metalloproteinase Activities

    SciTech Connect

    Liu, Guodong; Wang, Jun; Wunschel, David S.; Lin, Yuehe

    2006-09-27

    This communication describes a novel method for detecting of matrix metalloproteinase-7 activity using a peptide substrate labeled with a ferrocene reporter. The substrate serves as a selective ‘electrochemical proteolytic beacon’ (EPB) for this metalloproteinase. The EPB is immobilized on a gold electrode surface to enable ‘on-off’ electrochemical signaling capability for uncleaved and cleaved events. The EPB is efficiently and selectively cleaved by MMP-7 as measured by the rate of decrease in redox current of ferrocene. Direct transduction of a signal corresponding to peptide cleavage events into an electronic signal thus provides a simple, sensitive route for detecting the MMP activity. The new method allows for identification of the activity of MMP-7 in concentrations as low as 3.4 pM. The concept can be extended to design multiple peptide substrate labeled with different electroactive reporters for assaying multiple MMPs activities.

  20. Hyperosmotic Shock Engages Two Positive Feedback Loops through Caspase-3-dependent Proteolysis of JNK1-2 and Bid*

    PubMed Central

    Yue, Jicheng; Ben Messaoud, Nabil; López, José M.

    2015-01-01

    Hyperosmotic shock induces early calpain activation, Smac/DIABLO release from the mitochondria, and p38/JNK activation in Xenopus oocytes. These pathways regulate late cytochrome c release and caspase-3 activation. Here, we show that JNK1-1 and JNK1-2 are activated early by osmostress, and sustained activation of both isoforms accelerates the apoptotic program. When caspase-3 is activated, JNK1-2 is proteolyzed at Asp-385 increasing the release of cytochrome c and caspase-3 activity, thereby creating a positive feedback loop. Expression of Bcl-xL markedly reduces hyperosmotic shock-induced apoptosis. In contrast, expression of Bid induces rapid caspase-3 activation, even in the absence of osmostress, which is blocked by Bcl-xL co-expression. In these conditions a significant amount of Bid in the cytosol is mono- and bi-ubiquitinated. Caspase-3 activation by hyperosmotic shock induces proteolysis of Bid and mono-ubiquitinated Bid at Asp-52 increasing the release of cytochrome c and caspase-3 activation, and thus creating a second positive feedback loop. Revealing the JNK isoforms and the loops activated by osmostress could help to design better treatments for human diseases caused by perturbations in fluid osmolarity. PMID:26511318

  1. Hyperosmotic Shock Engages Two Positive Feedback Loops through Caspase-3-dependent Proteolysis of JNK1-2 and Bid.

    PubMed

    Yue, Jicheng; Ben Messaoud, Nabil; López, José M

    2015-12-18

    Hyperosmotic shock induces early calpain activation, Smac/DIABLO release from the mitochondria, and p38/JNK activation in Xenopus oocytes. These pathways regulate late cytochrome c release and caspase-3 activation. Here, we show that JNK1-1 and JNK1-2 are activated early by osmostress, and sustained activation of both isoforms accelerates the apoptotic program. When caspase-3 is activated, JNK1-2 is proteolyzed at Asp-385 increasing the release of cytochrome c and caspase-3 activity, thereby creating a positive feedback loop. Expression of Bcl-xL markedly reduces hyperosmotic shock-induced apoptosis. In contrast, expression of Bid induces rapid caspase-3 activation, even in the absence of osmostress, which is blocked by Bcl-xL co-expression. In these conditions a significant amount of Bid in the cytosol is mono- and bi-ubiquitinated. Caspase-3 activation by hyperosmotic shock induces proteolysis of Bid and mono-ubiquitinated Bid at Asp-52 increasing the release of cytochrome c and caspase-3 activation, and thus creating a second positive feedback loop. Revealing the JNK isoforms and the loops activated by osmostress could help to design better treatments for human diseases caused by perturbations in fluid osmolarity.

  2. Ellagic acid induces novel and atypical PKC isoforms and promotes caspase-3 dependent apoptosis by blocking energy metabolism.

    PubMed

    Mishra, Sudha; Vinayak, Manjula

    2014-01-01

    Antioxidant ellagic acid is a herbal polyphenolic compound shown to possess growth-inhibiting and apoptotic activities in cancer. Protein kinase C (PKC) plays an important role in cell proliferation, apoptosis, and differentiation. Apoptosis of tumor cells is induced by inactivation of glycolytic enzyme of anaerobic metabolism, lactate dehydrogenase (LDH)-A, and by activating apoptotic protein caspase-3 via PKCδ. The present study aims to analyze the role of ellagic acid on regulation of novel and atypical isozymes of PKC to modulate apoptosis and anaerobic metabolism to prevent lymphoma growth as its role on classical PKCs is reported earlier. Expression of novel and atypical isozymes of PKC, activity of PKCδ, expression and activity of caspase-3, and LDH-A have been analyzed. Expression is measured by RT-PCR, activities of PKCδ as level of its catalytic fragment, caspase-3 as level of its p17 fragment, and LDH-A by specific staining. Lymphoma bearing mice were treated with 3 different doses of ellagic acid. The treatment enhanced expression of all novel and atypical PKCs, activity and expression of caspase-3, and activity of PKCδ but decreased activity and expression of LDH-A. Our results suggest that ellagic acid induces apoptosis via novel and atypical PKCs in association with caspase-3 and induces cancer cell death by blocking the energy metabolism.

  3. Plasminogen-dependent proteolytic activity in Bifidobacterium lactis.

    PubMed

    Candela, Marco; Miccoli, Giacomo; Bergmann, Simone; Turroni, Silvia; Vitali, Beatrice; Hammerschmidt, Sven; Brigidi, Patrizia

    2008-08-01

    Bifidobacteria represent one of the most important health-promoting bacterial groups of the intestinal microbiota. The binding of plasminogen to species of Bifidobacterium has been recently reported. To further explore the interaction between bifidobacteria and plasminogen, we investigated the role of Bifidobacterium lactis BI07 plasminogen-dependent proteolytic activity in the degradation of host-specific substrates. Our experimental data demonstrate that the recruitment of plasminogen on the bacterial cell surface and its subsequent conversion into plasmin by host-derived plasminogen activators provide B. lactis BI07 with a surface-associated plasmin activity effective in degradation of physiological substrates such as extracellular matrix, fibronectin and fibrinogen. The ability of bifidobacteria to intervene in the host plasminogen/plasmin system may contribute to facilitating colonization of the host gastrointestinal tract.

  4. Aronia melanocarpa Juice Induces a Redox-Sensitive p73-Related Caspase 3-Dependent Apoptosis in Human Leukemia Cells

    PubMed Central

    Sharif, Tanveer; Alhosin, Mahmoud; Auger, Cyril; Minker, Carole; Kim, Jong-Hun; Etienne-Selloum, Nelly; Bories, Pierre; Gronemeyer, Hinrich; Lobstein, Annelise; Bronner, Christian; Fuhrmann, Guy; Schini-Kerth, Valérie B.

    2012-01-01

    Polyphenols are natural compounds widely present in fruits and vegetables, which have antimutagenic and anticancer properties. The aim of the present study was to determine the anticancer effect of a polyphenol-rich Aronia melanocarpa juice (AMJ) containing 7.15 g/L of polyphenols in the acute lymphoblastic leukemia Jurkat cell line, and, if so, to clarify the underlying mechanism and to identify the active polyphenols involved. AMJ inhibited cell proliferation, which was associated with cell cycle arrest in G2/M phase, and caused the induction of apoptosis. These effects were associated with an upregulation of the expression of tumor suppressor p73 and active caspase 3, and a downregulation of the expression of cyclin B1 and the epigenetic integrator UHRF1. AMJ significantly increased the formation of reactive oxygen species (ROS), decreased the mitochondrial membrane potential and caused the release of cytochrome c into the cytoplasm. Treatment with intracellular ROS scavengers prevented the AMJ-induced apoptosis and upregulation of the expression of p73 and active caspase 3. The fractionation of the AMJ and the use of identified isolated compounds indicated that the anticancer activity was associated predominantly with chlorogenic acids, some cyanidin glycosides, and derivatives of quercetin. AMJ treatment also induced apoptosis of different human lymphoblastic leukemia cells (HSB-2, Molt-4 and CCRF-CEM). In addition, AMJ exerted a strong pro-apoptotic effect in human primary lymphoblastic leukemia cells but not in human normal primary T-lymphocytes. Thus, the present findings indicate that AMJ exhibits strong anticancer activity through a redox-sensitive mechanism in the p53-deficient Jurkat cells and that this effect involves several types of polyphenols. They further suggest that AMJ has chemotherapeutic properties against acute lymphoblastic leukemia by selectively targeting lymphoblast-derived tumor cells. PMID:22412883

  5. Aronia melanocarpa juice induces a redox-sensitive p73-related caspase 3-dependent apoptosis in human leukemia cells.

    PubMed

    Sharif, Tanveer; Alhosin, Mahmoud; Auger, Cyril; Minker, Carole; Kim, Jong-Hun; Etienne-Selloum, Nelly; Bories, Pierre; Gronemeyer, Hinrich; Lobstein, Annelise; Bronner, Christian; Fuhrmann, Guy; Schini-Kerth, Valérie B

    2012-01-01

    Polyphenols are natural compounds widely present in fruits and vegetables, which have antimutagenic and anticancer properties. The aim of the present study was to determine the anticancer effect of a polyphenol-rich Aronia melanocarpa juice (AMJ) containing 7.15 g/L of polyphenols in the acute lymphoblastic leukemia Jurkat cell line, and, if so, to clarify the underlying mechanism and to identify the active polyphenols involved. AMJ inhibited cell proliferation, which was associated with cell cycle arrest in G(2)/M phase, and caused the induction of apoptosis. These effects were associated with an upregulation of the expression of tumor suppressor p73 and active caspase 3, and a downregulation of the expression of cyclin B1 and the epigenetic integrator UHRF1. AMJ significantly increased the formation of reactive oxygen species (ROS), decreased the mitochondrial membrane potential and caused the release of cytochrome c into the cytoplasm. Treatment with intracellular ROS scavengers prevented the AMJ-induced apoptosis and upregulation of the expression of p73 and active caspase 3. The fractionation of the AMJ and the use of identified isolated compounds indicated that the anticancer activity was associated predominantly with chlorogenic acids, some cyanidin glycosides, and derivatives of quercetin. AMJ treatment also induced apoptosis of different human lymphoblastic leukemia cells (HSB-2, Molt-4 and CCRF-CEM). In addition, AMJ exerted a strong pro-apoptotic effect in human primary lymphoblastic leukemia cells but not in human normal primary T-lymphocytes. Thus, the present findings indicate that AMJ exhibits strong anticancer activity through a redox-sensitive mechanism in the p53-deficient Jurkat cells and that this effect involves several types of polyphenols. They further suggest that AMJ has chemotherapeutic properties against acute lymphoblastic leukemia by selectively targeting lymphoblast-derived tumor cells.

  6. Morus alba Accumulates Reactive Oxygen Species to Initiate Apoptosis via FOXO-Caspase 3-Dependent Pathway in Neuroblastoma Cells

    PubMed Central

    Kwon, Young Hwi; Bishayee, Kausik; Rahman, Ataur; Hong, Jae Seung; Lim, Soon-Sung; Huh, Sung-Oh

    2015-01-01

    Morus alba root extract (MARE) has been used to treat hyperglycaemic conditions in oriental medicine. Here, we studied whether MARE possesses a cytotoxic effect on neuroblastoma. To check the cytotoxicity generated by MARE was whether relatively higher against the cancer cells rather than normal cells, we chose a neuroblastoma cell line (B103) and a normal cell line (Rat-2). A CCK assay revealed that MARE (10 μg/ml) reduced cell viability to approximately 60% compared to an untreated control in B103 cells. But in Rat-2 cells, MARE induced relatively lower cytotoxicity. To investigate the mechanisms underlying the cytotoxic effect of MARE, we used flow cytometry combined with immunoblot analyses. We found that MARE-treatment could accumulate ROS and depolarize mitochondria membrane potential of B103 cells. Further treatment with MARE in B103 cells also could damage DNA and induce apoptosis. An expression study of p-Akt also suggested that there was a reduction in cellular proliferation and transcription along with the process of apoptosis, which was further evidenced by an increase in Bax and cleaved-caspase 3 activity. Together, our findings suggest that MARE produces more cytotoxicity in cancer cells while having a relatively attenuated effect on normal cells. As such, MARE may be a safer option in cancer therapeutics, and it also shows potential for the patients with symptoms of hyperglycemia and cancer. PMID:25921607

  7. Dopaminergic neurotoxicant 6-OHDA induces oxidative damage through proteolytic activation of PKC{delta} in cell culture and animal models of Parkinson's disease

    SciTech Connect

    Latchoumycandane, Calivarathan; Anantharam, Vellareddy; Jin, Huajun; Kanthasamy, Anumantha; Kanthasamy, Arthi

    2011-11-15

    The neurotoxicant 6-hydroxydopamine (6-OHDA) is used to investigate the cellular and molecular mechanisms underlying selective degeneration of dopaminergic neurons in Parkinson's disease (PD). Oxidative stress and caspase activation contribute to the 6-OHDA-induced apoptotic cell death of dopaminergic neurons. In the present study, we sought to systematically characterize the key downstream signaling molecule involved in 6-OHDA-induced dopaminergic degeneration in cell culture and animal models of PD. Treatment of mesencephalic dopaminergic neuronal N27 cells with 6-OHDA (100 {mu}M) for 24 h significantly reduced mitochondrial activity and increased cytosolic cytochrome c, followed by sequential activation of caspase-9 and caspase-3. Co-treatment with the free radical scavenger MnTBAP (10 {mu}M) significantly attenuated 6-OHDA-induced caspase activities. Interestingly, 6-OHDA induced proteolytic cleavage and activation of protein kinase C delta (PKC{delta}) was completely suppressed by treatment with a caspase-3-specific inhibitor, Z-DEVD-FMK (50 {mu}M). Furthermore, expression of caspase-3 cleavage site-resistant mutant PKC{delta}{sup D327A} and kinase dead PKC{delta}{sup K376R} or siRNA-mediated knockdown of PKC{delta} protected against 6-OHDA-induced neuronal cell death, suggesting that caspase-3-dependent PKC{delta} promotes oxidative stress-induced dopaminergic degeneration. Suppression of PKC{delta} expression by siRNA also effectively protected N27 cells from 6-OHDA-induced apoptotic cell death. PKC{delta} cleavage was also observed in the substantia nigra of 6-OHDA-injected C57 black mice but not in control animals. Viral-mediated delivery of PKC{delta}{sup D327A} protein protected against 6-OHDA-induced PKC{delta} activation in mouse substantia nigra. Collectively, these results strongly suggest that proteolytic activation of PKC{delta} is a key downstream event in dopaminergic degeneration, and these results may have important translational value for

  8. Detecting kallikrein proteolytic activity with peptide-quantum dot nanosensors.

    PubMed

    Breger, Joyce C; Sapsford, Kim E; Ganek, Jessica; Susumu, Kimihiro; Stewart, Michael H; Medintz, Igor L

    2014-07-23

    Contamination and adulterants in both naturally derived and synthetic drugs pose a serious threat to the worldwide medical community. Developing rapid and sensitive sensors/devices to detect these hazards is thus a continuing need. We describe a hydrophilic semiconductor quantum dot (QD)-peptide Förster resonance energy transfer (FRET) nanosensor for monitoring the activity of kallikrein, a key proteolytic enzyme functioning at the initiation of the blood clotting cascade. Kallikrein is also activated by the presence of an oversulfated contaminant recently found in preparations of the drug heparin. Quantitatively monitoring the activity of this enzyme within a nanosensor format has proven challenging because of inherent steric and kinetic considerations. Our sensor is designed around a central QD donor platform which displays controlled ratios of a modular peptidyl substrate. This peptide, in turn, sequentially expresses a terminal oligohistidine motif that mediates the rapid self-assembly of peptides to the QD surface, a linker-spacer sequence to extend the peptide away from the QD surface, a kallikrein recognized-cleavage site, and terminates in an acceptor dye-labeling site. Hydrophilic QDs prepared with compact, zwitterionic surface coatings were first evaluated for their ability to self-assemble the dye-labeled peptide substrates. An optimized two-step protocol was then utilized where high concentrations of peptide were initially digested with purified human kallikrein and samples collected at distinct time points were subsequently diluted into QD-containing solutions for assaying. This sensor provided a quantitative FRET-based readout for monitoring kallikrein activity and comparison to a calibration curve allowed estimation of the relevant Michaelis-Menten kinetic descriptors. The results further suggest that almost any protease should be amenable to a QD-based FRET assay format with appropriate design considerations.

  9. Substrate supply, fine roots, and temperature control proteolytic enzyme activity in temperate forest soils.

    PubMed

    Brzostek, Edward R; Finzi, Adrien C

    2011-04-01

    Temperature and substrate availability constrain the activity of the extracellular enzymes that decompose and release nutrients from soil organic matter (SOM). Proteolytic enzymes are the primary class of enzymes involved in the depolymerization of nitrogen (N) from proteinaceous components of SOM, and their activity affects the rate of N cycling in forest soils. The objectives of this study were to determine whether and how temperature and substrate availability affect the activity of proteolytic enzymes in temperate forest soils, and whether the activity of proteolytic enzymes and other enzymes involved in the acquisition of N (i.e., chitinolytic and ligninolytic enzymes) differs between trees species that form associations with either ectomycorrhizal or arbuscular mycorrhizal fungi. Temperature limitation of proteolytic enzyme activity was observed only early in the growing season when soil temperatures in the field were near 4 degrees C. Substrate limitation to proteolytic activity persisted well into the growing season. Ligninolytic enzyme activity was higher in soils dominated by ectomycorrhizal associated tree species. In contrast, the activity of proteolytic and chitinolytic enzymes did not differ, but there were differences between mycorrhizal association in the control of roots on enzyme activity. Roots of ectomycorrhizal species but not those of arbuscular mycorrhizal species exerted significant control over proteolytic, chitinolytic, and ligninolytic enzyme activity; the absence of ectomycorrhizal fine roots reduced the activity of all three enzymes. These results suggest that climate warming in the absence of increases in substrate availability may have a modest effect on soil-N cycling, and that global changes that alter belowground carbon allocation by trees are likely to have a larger effect on nitrogen cycling in stands dominated by ectomycorrhizal fungi.

  10. Proteolytic activity and immunogenicity of oral bromelain within the gastrointestinal tract of mice.

    PubMed

    Hale, Laura P

    2004-02-01

    Bromelain is a mixture of proteinases derived from pineapple stem that is marketed by health food stores as a "digestive aid". A number of studies suggest that bromelain may also have anti-inflammatory activity in vivo, including an anecdotal report describing potential efficacy in inflammatory bowel disease. We and others have previously shown that proteolytically active bromelain removes certain cell surface molecules and affects leukocyte migration, activation, and production of cytokines and inflammatory mediators in vitro. The purpose of this study was to determine whether ingested bromelain retains proteolytic activity within the murine gastrointestinal tract in vivo. The proteolytic activity of bromelain was determined in vitro using model substrates or immunofluorescence assays after administration of various doses and formulations orally to mice. Immune responses against bromelain were detected by enzyme immunoassays. When formulated in antacid, oral bromelain retained substantial proteolytic activity throughout the gastrointestinal tract. Bromelain concentrations within the colon were dependent on both dose and formulation and were sufficient to remove bromelain-sensitive molecules from both leukocytes and colon epithelial cells. Peak activity in the stool was observed 4 h after oral dosing. Although anti-bromelain IgG was detected in both serum and stool after long-term oral therapy, these antibodies did not prevent bromelain proteolytic activity within the gastrointestinal tract. These studies demonstrate that bromelain enzymes can remain intact and proteolytically active within the murine gastrointestinal tract. They provide further support for the hypothesis that oral bromelain may potentially modify inflammation within the gastrointestinal tract via local proteolytic activity within the colonic microenvironment.

  11. T-cell receptor-induced JNK activation requires proteolytic inactivation of CYLD by MALT1.

    PubMed

    Staal, Jens; Driege, Yasmine; Bekaert, Tine; Demeyer, Annelies; Muyllaert, David; Van Damme, Petra; Gevaert, Kris; Beyaert, Rudi

    2011-05-04

    The paracaspase mucosa-associated lymphoid tissue 1 (MALT1) is central to lymphocyte activation and lymphomagenesis. MALT1 mediates antigen receptor signalling to NF-κB by acting as a scaffold protein. Furthermore, MALT1 has proteolytic activity that contributes to optimal NF-κB activation by cleaving the NF-κB inhibitor A20. Whether MALT1 protease activity is involved in other signalling pathways, and the identity of the relevant substrates, is unknown. Here, we show that T-cell receptors (TCR) activation, as well as overexpression of the oncogenic API2-MALT1 fusion protein, results in proteolytic inactivation of CYLD by MALT1, which is specifically required for c-jun N-terminal kinase (JNK) activation and the inducible expression of a subset of genes. These results indicate a novel role for MALT1 proteolytic activity in TCR-induced JNK activation and reveal CYLD cleavage as the underlying mechanism.

  12. Partial purification of histone H3 proteolytic activity from the budding yeast Saccharomyces cerevisiae.

    PubMed

    Azad, Gajendra Kumar; Tomar, Raghuvir Singh

    2016-06-01

    The proteolytic clipping of histone tails has recently emerged as a novel form of irreversible post-translational modification (PTM) of histones. Histone clipping has been implicated as a regulatory process leading to the permanent removal of PTMs from histone proteins. However, there is scarcity of literature that describes the identification and characterization of histone-specific proteases. Here, we employed various biochemical methods to report histone H3-specific proteolytic activity from budding yeast. Our results demonstrate that H3 proteolytic activity was associated with sepharose bead matrices and activity was not affected by a variety of stress conditions. We have also identified the existence of an unknown protein that acts as a physiological inhibitor of the H3-clipping activity of yeast H3 protease. Moreover, through protease inhibition assays, we have also characterized yeast H3 protease as a serine protease. Interestingly, unlike glutamate dehydrogenase (GDH), yeast H3 proteolytic activity was not inhibited by Stefin B. Together, our findings suggest the existence of a novel H3 protease in yeast that is different from other reported histone H3 proteases. The presence of histone H3 proteolytic activity, along with the physiological inhibitor in yeast, suggests an interesting molecular mechanism that regulates the activity of histone proteases. Copyright © 2016 John Wiley & Sons, Ltd.

  13. Proteolytic activation of protein kinase C delta by an ICE-like protease in apoptotic cells.

    PubMed Central

    Emoto, Y; Manome, Y; Meinhardt, G; Kisaki, H; Kharbanda, S; Robertson, M; Ghayur, T; Wong, W W; Kamen, R; Weichselbaum, R

    1995-01-01

    These studies demonstrate that treatment of human U-937 cells with ionizing radiation (IR) is associated with activation of a cytoplasmic myelin basic protein (MBP) kinase. Characterization of the kinase by gel filtration and in-gel kinase assays support activation of a 40 kDa protein. Substrate and inhibitor studies further support the induction of protein kinase C (PKC)-like activity. The results of N-terminal amino acid sequencing of the purified protein demonstrate identity of the kinase with an internal region of PKC delta. Immunoblot analysis was used to confirm proteolytic cleavage of intact 78 kDa PKC delta in control cells to the 40 kDa C-terminal fragment after IR exposure. The finding that both IR-induced proteolytic activation of PKC delta and endonucleolytic DNA fragmentation are blocked by Bcl-2 and Bcl-xL supports an association with physiological cell death (PCD). Moreover, cleavage of PKC delta occurs adjacent to aspartic acid at a site (QDN) similar to that involved in proteolytic activation of interleukin-1 beta converting enzyme (ICE). The specific tetrapeptide ICE inhibitor (YVAD) blocked both proteolytic activation of PKC delta and internucleosomal DNA fragmentation in IR-treated cells. These findings demonstrate that PCD is associated with proteolytic activation of PKC delta by an ICE-like protease. Images PMID:8557034

  14. Proteolytic Activity Matrix Analysis (PrAMA) for Simultaneous Determination of Multiple Protease Activities

    PubMed Central

    Miller, Miles A.; Barkal, Layla; Jeng, Karen; Herrlich, Andreas; Griffith, Linda G.; Lauffenburger, Douglas A.

    2011-01-01

    Matrix metalloproteinases (MMPs) and A Disintegrin and Metalloproteinases (ADAMs) are two related protease families that play key roles in matrix remodeling and growth factor ligand shedding. Directly ascertaining the proteolytic activities of particular MMPs and ADAMs in physiological environments in a non-invasive, real-time, multiplex manner remains a challenge. This work describes Proteolytic Activity Matrix Analysis (PrAMA), an integrated experimental measurement and mathematical analysis framework for simultaneously determining the activities of particular enzymes in complex mixtures of MMPs and ADAMs. The PrAMA method interprets dynamic signals from panels of moderately specific FRET-based polypeptide protease substrates to deduce a profile of specific MMP and ADAM proteolytic activities. Deconvolution of signals from complex mixtures of proteases is accomplished using prior data on individual MMP/ADAM cleavage signatures for the substrate panel measured with purified enzymes. We first validate PrAMA inference using a compendium of roughly 4000 measurements involving known mixtures of purified enzymes and substrates, and then demonstrate application to the live-cell response of wildtype, ADAM10−/−, and ADAM17−/− fibroblasts to phorbol ester stimulation. Results indicate PrAMA can distinguish closely related enzymes from each other with high accuracy, even in the presence of unknown background proteolytic activity. PrAMA offers a valuable tool for applications ranging from live-cell in vitro assays to high-throughput inhibitor screening with complex enzyme mixtures. Moreover, our approach may extend to other families of proteases, such as caspases and cathepsins, that also can lack highly-specific substrates. PMID:21180771

  15. An examination of the proteolytic activity for bovine pregnancy-associated glycoproteins 2 and 12.

    PubMed

    Telugu, Bhanu Prakash V L; Palmier, Mark O; Van Doren, Steven R; Green, Jonathan A

    2010-01-01

    The pregnancy-associated glycoproteins (PAGs) represent a complex group of putative aspartic peptidases expressed exclusively in the placentas of species in the Artiodactyla order. The ruminant PAGs segregate into two classes: the 'ancient' and 'modern' PAGs. Some of the modern PAGs possess alterations in the catalytic center that are predicted to preclude their ability to act as peptidases. The ancient ruminant PAGs in contrast are thought to be peptidases, although no proteolytic activity has been described for these members. The aim of the present study was to investigate (1) if the ancient bovine PAGs (PAG-2 and PAG-12) have proteolytic activity, and (2) if there are any differences in activity between these two closely related members. Recombinant bovine PAG-2 and PAG-12 were expressed in a baculovirus expression system and the purified proteins were analyzed for proteolytic activity against a synthetic fluorescent cathepsin D/E substrate. Both proteins exhibited proteolytic activity with acidic pH optima. The k(cat)/K(m) for bovine PAG-2 was 2.7x10(5) m(-1) s(-1) and for boPAG-12 it was 6.8x10(4) m(-1) s(-1). The enzymes were inhibited by pepstatin A with a K(i) of 0.56 and 7.5 nm for boPAG-2 and boPAG-12, respectively. This is the first report describing proteolytic activity in PAGs from ruminant ungulates.

  16. An Examination of the Proteolytic Activity for Bovine Pregnancy-Associated Glycoprotein 2 and 12

    PubMed Central

    Telugu, Bhanu Prakash V.L.; Palmier, Mark O.; Van Doren, Steven R.; Green, Jonathan A.

    2010-01-01

    The pregnancy-associated glycoproteins (PAGs) represent a complex group of putative aspartic peptidases expressed exclusively in the placentas of species in the Artiodactyla order. The ruminant PAGs segregate into two classes -the ‘ancient’ and ‘modern’ PAGs. Some of the modern PAGs possess alterations in the catalytic center that are predicted to preclude their ability to act as peptidases. The ancient ruminant PAGs in contrast are thought to be peptidases, although, no proteolytic activity has been described for these members. The goal of this present study was to investigate (1) if the ancient bovine PAGs (PAGs-2 and -12) have proteolytic activity, and (2) if there are any differences in activity between these two closely related members. Recombinant bovine PAGs-2 and -12 were expressed in a baculovirus expression system and the purified proteins were analyzed for proteolytic activity against a synthetic fluorescent cathepsin D/E substrate. Both proteins exhibited proteolytic activity with acidic pH optima. The kcat/KM for bovine PAG-2 was 2.7×105 M−1s−1 and for boPAG-12 it was 6.8×104 M−1s−1. The enzymes were inhibited by pepstatin A with a Ki of 0.56 and 7.5 nM for boPAG-2 and boPAG-12, respectively. This is the first report describing proteolytic activity in PAGs from ruminant ungulates. PMID:20030586

  17. Increase in larval gut proteolytic activities and Bti resistance in the Dengue fever mosquito.

    PubMed

    Tetreau, Guillaume; Stalinski, Renaud; David, Jean-Philippe; Després, Laurence

    2013-02-01

    The bioinsecticide Bacillus thuringiensis var. israelensis (Bti) is increasingly used worldwide for mosquito control. Although no established resistance to Bti has been described in the field so far, a resistant Aedes aegypti strain (LiTOX strain) was selected in the laboratory using field-collected leaf litter containing Bti toxins. This selected strain exhibits a moderate resistance level to Bti, but a high resistance level to individual Cry toxins. As Bti contains four different toxins, generalist resistance mechanisms affecting mosquito tolerance to different toxins were expected in the resistant strain. In the present work, we show that the resistant strain exhibits an increase of various gut proteolytic activities including trypsins, leucine-aminopeptidases, and carboxypeptidase A activities. These elevated proteolytic activities resulted in a faster activation of Cry4Aa protoxins while Cry4Ba or Cry11Aa were not affected. These results suggest that changes in proteolytic activities may contribute to Bti resistance in mosquitoes together with other mechanisms.

  18. Near Infrared Optical Proteolytic Beacons for In Vivo Imaging of Matrix Metalloproteinase Activity

    PubMed Central

    McIntyre, J. Oliver; Scherer, Randy L.; Matrisian, Lynn M.

    2010-01-01

    The exuberant expression of proteinases by tumor cells has long been associated with the breakdown of the extracellular matrix, tumor invasion, and metastasis to distant organs. There is both epidemiological and experimental data that support a causative role for proteinases of the matrix metalloproteinase (MMP) family in tumor progression. Optical imaging techniques provide an extraordinary opportunity for non-invasive “molecular imaging” of tumor-associated proteolytic activity. The application of optical proteolytic beacons for the detection of specific proteinase activities associated with tumors has several potential purposes: 1) Detection of small, early-stage tumors with increased sensitivity due to the catalytic nature of proteolytic activity, 2) Diagnosis and Prognosis to distinguished tumors that require particularly aggressive therapy or those that will not benefit from therapy, 3) Identification of tumors appropriate for specific anti-proteinase therapeutics and optimization of drug and dose based on determination of target modulation, and 4) as an indicator of efficacy of proteolytically-activated pro-drugs. This chapter describes the synthesis, characterization, and application of reagents that use visible and near infrared fluorescence resonance energy transfer (FRET) fluorophore pairs to detect and measure MMP-referable proteolytic activity in tumors in mouse models of cancer. PMID:20135290

  19. Effects of Geroprotectors on Age-Related Changes in Proteolytic Digestive Enzyme Activities at Different Lighting Conditions.

    PubMed

    Morozov, A V; Khizhkin, E A; Svechkina, E B; Vinogradova, I A; Ilyukha, V A; Anisimov, V N; Khavinson, V Kh

    2015-10-01

    We studied the effect of melatonin and epithalon on age-related changes in proteolytic digestive enzyme activity in the pancreas and gastric mucosa of rats kept under different lighting conditions. In rats kept under standard illumination, pepsin activity and the total proteolytic activity in the stomach and pancreas increased by the age of 12 months, but then decreased. Constant and natural lighting disturbed the age dynamics of proteolytic digestive enzyme activity. Administration of melatonin and epithalon to animals exposed to constant lighting restored age dynamics of pepsin activity and little affected total proteolytic activity.

  20. [Proteolytic activity of IgG-antibodies of mice, immunized by calf thymus histones].

    PubMed

    Kit, Iu Ia; Korniĭ, N; Kril', I Ĭ; Mahorivs'ka, I B; Tkachenko, V; Bilyĭ, R O; Stoĭka, R S

    2014-01-01

    The main goal of the study was to determine the ability of histones to induce production of the proteolytically active IgG-antibodies in BALB/c mice. In order to perform this study 8 mice were immunized with the fraction of total calf thymus histones. IgGs were isolated from the serum of the immunized and not immunized animals by means of precipitation with 33% ammonium sulfate, followed by affinity chromatography on protein G-Sepharose column. Histones, myelin basic protein (MBP), lysozyme, BSA, ovalbumin, macroglobulin, casein and cytochrome c served as substrates for determining the proteolytic activity. It was found that IgGs from the blood serum of immunized mice are capable of hydrolyzing histone H1, core histone and MBP. On the contrary, the proteolytic activity of IgGs from the blood serum of not immunized mice was not detected. The absence of proteolytical enzymes in the fraction of IgGs was proven by HPLC chromatography. High levels of proteolytic activity toward histones have been also detected in affinity purified IgGs from blood serum of patients with rheumatoid arthritis, but not in healthy donors. These data indicate that eukaryotic histones may induce production of protabzymes in mammals. The possible origin of these protabzymes and their potential biological role in mammalians is discussed.

  1. Seasonal variation in the temperature sensitivity of proteolytic enzyme activity in temperate forest soils

    NASA Astrophysics Data System (ADS)

    Brzostek, Edward R.; Finzi, Adrien C.

    2012-03-01

    Increasing soil temperature has the potential to alter the activity of the extracellular enzymes that mobilize nitrogen (N) from soil organic matter (SOM) and ultimately the availability of N for primary production. Proteolytic enzymes depolymerize N from proteinaceous components of SOM into amino acids, and their activity is a principal driver of the within-system cycle of soil N. The objectives of this study were to investigate whether the soils of temperate forest tree species differ in the temperature sensitivity of proteolytic enzyme activity over the growing season and the role of substrate limitation in regulating temperature sensitivity. Across species and sampling dates, proteolytic enzyme activity had relatively low sensitivity to temperature with a mean activation energy (Ea) of 33.5 kJ mol-1. Ea declined in white ash, American beech, and eastern hemlock soils across the growing season as soils warmed. By contrast, Eain sugar maple soil increased across the growing season. We used these data to develop a species-specific empirical model of proteolytic enzyme activity for the 2009 calendar year and studied the interactive effects of soil temperature (ambient or +5°C) and substrate limitation (ambient or elevated protein) on enzyme activity. Declines in substrate limitation had a larger single-factor effect on proteolytic enzyme activity than temperature, particularly in the spring. There was, however, a large synergistic effect of increasing temperature and substrate supply on proteolytic enzyme activity. Our results suggest limited increases in N availability with climate warming unless there is a parallel increase in the availability of protein substrates.

  2. Proteolytic activity of the plum pox potyvirus NIa-like protein in Escherichia coli.

    PubMed

    García, J A; Riechmann, J L; Laín, S

    1989-06-01

    The nucleotide sequence of the small nuclear inclusion protein (NIa)-like cistron of plum pox potyvirus (PPV) has been determined. Viral proteolytic activity was expressed in Escherichia coli cells harboring plasmids with a PPV cDNA insert approximately 7000 nt long. Free PPV capsid protein was detected in these cells, but it was not produced when a mutation was introduced in the PPV cDNA insert which induced a Gln to Pro substitution at the large nuclear inclusion protein (NIb)-capsid protein junction. By mutational analysis, the NIa-like protein was determined to be responsible for the proteolytic activity. A Gln to Ser substitution at the presumed NIa-NIb junction, which inhibited proteolytic processing at the carboxyl end of the protease, had no effect on proteolytic cleavage at the NIb-capsid protein junction. In contrast with the high efficiency of proteolytic processing at the NIb-capsid protein cleavage site, processing at the ends of the PPV protease was not complete, suggesting that the PPV polyprotein, like that of other potyviruses, contains cleavage sites with different properties.

  3. Proteolytic activity in Fasciola hepatica is reduced by the administration of cathepsin L mimotopes.

    PubMed

    Villa-Mancera, A; Quiroz-Romero, H; Correa, D; Alonso, R A

    2011-03-01

    The objective of this study was to assess the proteolytic activity of Fasciola hepatica cathepsins in liver sections from mice vaccinated with phage clones of cathepsin L mimotopes, using the film in situ zymography technique. Female BALB/c mice were immunized three times with 2.5 x 10¹¹ phage particles without adjuvant. Animals vaccinated with phage clones produced high titres of anti-mimotope antibodies and a significant reduction in fluke burden was observed following challenge with metacercariae of F. hepatica. The proteolytic activity in hepatic tissue was reduced after the immunization with phage clones.

  4. Neuropathogenic Escherichia coli K1 does not exhibit proteolytic activities to exert its pathogenicity

    PubMed Central

    2013-01-01

    Background Proteases are well-known virulence factors that promote survival, pathogenesis and immune evasion of many pathogens. Several lines of evidence suggest that the blood–brain barrier permeability is a prerequisite in microbial invasion of the central nervous system. Because proteases are frequently associated with vascular permeability by targeting junctional proteins, here it is hypothesized that neuropathogenic Escherichia coli K1 exhibit proteolytic activities to exert its pathogenicity. Methods Zymographic assays were performed using collagen and gelatin as substrates. The lysates of whole E. coli K1 strain E44, or E. coli K-12 strain HB101 were tested for proteolytic activities. The conditioned media were prepared by incubating bacteria in RPMI-1640 in the presence or absence of serum. The cell-free supernatants were collected and tested for proteases in zymography as mentioned above. Additionally, proteolytic degradation of host immune factors was determined by co-incubating conditioned media with albumin/immunoglobulins using protease assays. Results When collagen or gelatin were used as substrates in zymographic assays, neither whole bacteria nor conditioned media exhibited proteolytic activities. The conditioned media of neuropathogenic E. coli K1 strain E44, or E. coli K-12 strain HB101 did not affect degradation of albumin and immunoglobulins using protease assays. Conclusions Neither zymographic assays nor protease assays detected proteolytic activities in either the whole bacteria or conditioned media of E. coli K1 strain E44 and E. coli K-12 strain HB101. These findings suggest that host cell monolayer disruptions and immune evasion strategies are likely independent of proteolytic activities of neuropathogenic E. coli K1. PMID:23634997

  5. Proteolytic activity and cytokine up-regulation by non-albicans Candida albicans.

    PubMed

    Nawaz, Ali; Pärnänen, Pirjo; Kari, Kirsti; Meurman, Jukka H

    2015-05-01

    Mouth is an important source of infections and oral infections such as Candida infections increase the risk of mortality. Our purpose was to investigate differences in proteolytic activity of non-albicans Candida albicans (non-albicans Candida) between clinical isolates and laboratory samples. The second aim was to assess the concentration of pro- and anti-inflammatory cytokine levels IL-1β, IL-10, and TNF-α in saliva of patients with the non-albicans Candida and Candida-negative saliva samples. Clinical yeast samples from our laboratory were used for analyses. Candida strains were grown in YPG at 37 °C for 24 h in water bath with shaking. The activity of Candida proteinases of cell and cell-free fractions were analyzed by MDPF-gelatin zymography. The levels of IL-1β, IL-10, and TNF-α were measured from saliva with ELISA. The study showed differences in the proteolytic activity among the non-albicans Candida strains. C. tropicalis had higher proteolytic activity when compared to the other strains. Significant difference was found in salivary IL-1β levels between the non-albicans Candida and control strains (P < 0.002). The present findings showed differences in proteolytic activity among the non-albicans Candida strains. The increased IL-1β concentration may be one of the host response components associated with non-albicans Candida infection.

  6. Proteolytic and antimicrobial activity of lactic acid bacteria grown in goat milk.

    PubMed

    Atanasova, Jivka; Moncheva, Penka; Ivanova, Iskra

    2014-11-02

    We examined 62 strains and 21 trade starter cultures from the collection of LB Bulgaricum PLC for proteolytic and antimicrobial activity of lactic acid bacteria (LAB) grown in goat milk. The aim of this study was to investigate the fermentation of caseins, α-lactalbumin and β-lactoglobulin by LAB, using the o-phthaldialdehyde (OPA) spectrophotometric assay and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The proteolysis targeted mainly caseins, especially β-casein. Whey proteins were proteolyzed, essentially β-lactoglobulin. The proteolytic activity of Lactococcus lactis l598, Streptococcus thermophilus t3D1, Dt1, Lactobacillus lactis 1043 and L. delbrueckii subsp. bulgaricus b38, b122 and b24 was notably high. The proteolysis process gave rise to medium-sized peptide populations. Most of the examined strains showed antimicrobial activity against some food pathogens, such as Escherichia coli, Staphylococcus aureus, Salmonella cholere enteridis, Listeria monocytogenes, Listeria innocua and Enterobacter aerogenes. The most active producers of antimicrobial-active peptides were strains of L. delbrueckii subsp. bulgaricus and S. thermophilus, which are of practical importance. The starter cultures containing the examined species showed high proteolytic and antimicrobial activity in skimmed goat milk. The greatest antimicrobial activity of the cultures was detected against E. aerogenes. The obtained results demonstrated the significant proteolytic potential of the examined strains in goat milk and their potential for application in the production of dairy products from goat's milk. The present results could be considered as the first data on the proteolytic capacity of strains and starter cultures in goat milk for the purposes of trade interest of LB Bulgaricum PLC.

  7. Proteolytic activity of IgGs from blood serum of Wistar rats at experimental rheumatoid arthritis.

    PubMed

    Kit, Yu Ya; Myronovsky, S L; Kril, I I; Havrylyuk, A M; Chop'yak, V V; Stoika, R S

    2014-01-01

    The aim of this work was to study the proteolytic activity of IgGs purified from blood serum of Wistar rats at experimental rheumatoid arthritis (ERA) induced by an injection of bovine collagen of type II. Twenty rats were immunized with a preparation of bovine collagen II (Sigma-Aldrich, USA) in the presence of complete Freund's adjuvant. ERA development was determined by inflammation in limbs of treated animals. IgG preparations were isolated from blood serum of immunized and non-immunized animals by precipitation of antibodies with 33% ammonium sulfate followed by chromatography on the Protein G-Sepharose column. Human histone H1, bovine collagen II, calf thymus histones, myelin basic protein (MBP), bovine serum albumin (BSA), and bovine casein were used as substrates of the proteolytic activity of IgGs. It was found that IgG preparations from blood serum of rats with ERA were capable of cleaving histone H1 and MBP, however, they were catalytically inactive towards collagen II, casein, BSA, and core histones. IgGs from blood serum of non-immunized rats were proteolytically inactive towards all used protein substrates. Thus, we demonstrated that immunization of rats with bovine collagen II induced IgG-antibodies possessing the proteolytic activity towards histone H1 and MBP. This activity might be associated with the development of inflammatory processes in the immunized rats.

  8. Discovery of a proteolytic flagellin family in diverse bacterial phyla that assembles enzymatically active flagella.

    PubMed

    Eckhard, Ulrich; Bandukwala, Hina; Mansfield, Michael J; Marino, Giada; Cheng, Jiujun; Wallace, Iain; Holyoak, Todd; Charles, Trevor C; Austin, John; Overall, Christopher M; Doxey, Andrew C

    2017-09-12

    Bacterial flagella are cell locomotion and occasional adhesion organelles composed primarily of the polymeric protein flagellin, but to date have not been associated with any enzymatic function. Here, we report the bioinformatics-driven discovery of a class of enzymatic flagellins that assemble to form proteolytically active flagella. Originating by a metallopeptidase insertion into the central flagellin hypervariable region, this flagellin family has expanded to at least 74 bacterial species. In the pathogen, Clostridium haemolyticum, metallopeptidase-containing flagellin (which we termed flagellinolysin) is the second most abundant protein in the flagella and is localized to the extracellular flagellar surface. Purified flagellar filaments and recombinant flagellin exhibit proteolytic activity, cleaving nearly 1000 different peptides. With ~ 20,000 flagellin copies per  ~ 10-μm flagella this assembles the largest proteolytic complex known. Flagellum-mediated extracellular proteolysis expands our understanding of the functional plasticity of bacterial flagella, revealing this family as enzymatic biopolymers that mediate interactions with diverse peptide substrates.So far no enzymatic activity has been attributed to flagellin, the major component of bacterial flagella. Here the authors use bioinformatic analysis and identify a metallopeptidase insertion in flagellins from 74 bacterial species and show that recombinant flagellin and flagellar filaments have proteolytic activity.

  9. Glucocorticoids activate the ATP-ubiquitin-dependent proteolytic system in skeletal muscle during fasting

    NASA Technical Reports Server (NTRS)

    Wing, S. S.; Goldberg, A. L.; Goldberger, A. L. (Principal Investigator)

    1993-01-01

    Glucocorticoids are essential for the increase in protein breakdown in skeletal muscle normally seen during fasting. To determine which proteolytic pathway(s) are activated upon fasting, leg muscles from fed and fasted normal rats were incubated under conditions that block or activate different proteolytic systems. After food deprivation (1 day), the nonlysosomal ATP-dependent process increased by 250%, as shown in experiments involving depletion of muscle ATP. Also, the maximal capacity of the lysosomal process increased 60-100%, but no changes occurred in the Ca(2+)-dependent or the residual energy-independent proteolytic processes. In muscles from fasted normal and adrenalectomized (ADX) rats, the protein breakdown sensitive to inhibitors of the lysosomal or Ca(2+)-dependent pathways did not differ. However, the ATP-dependent process was 30% slower in muscles from fasted ADX rats. Administering dexamethasone to these animals or incubating their muscles with dexamethasone reversed this defect. During fasting, when the ATP-dependent process rises, muscles show a two- to threefold increase in levels of ubiquitin (Ub) mRNA. However, muscles of ADX animals failed to show this response. Injecting dexamethasone into the fasted ADX animals increased muscle Ub mRNA within 6 h. Thus glucocorticoids activate the ATP-Ub-dependent proteolytic pathway in fasting apparently by enhancing the expression of components of this system such as Ub.

  10. Glucocorticoids activate the ATP-ubiquitin-dependent proteolytic system in skeletal muscle during fasting

    NASA Technical Reports Server (NTRS)

    Wing, S. S.; Goldberg, A. L.; Goldberger, A. L. (Principal Investigator)

    1993-01-01

    Glucocorticoids are essential for the increase in protein breakdown in skeletal muscle normally seen during fasting. To determine which proteolytic pathway(s) are activated upon fasting, leg muscles from fed and fasted normal rats were incubated under conditions that block or activate different proteolytic systems. After food deprivation (1 day), the nonlysosomal ATP-dependent process increased by 250%, as shown in experiments involving depletion of muscle ATP. Also, the maximal capacity of the lysosomal process increased 60-100%, but no changes occurred in the Ca(2+)-dependent or the residual energy-independent proteolytic processes. In muscles from fasted normal and adrenalectomized (ADX) rats, the protein breakdown sensitive to inhibitors of the lysosomal or Ca(2+)-dependent pathways did not differ. However, the ATP-dependent process was 30% slower in muscles from fasted ADX rats. Administering dexamethasone to these animals or incubating their muscles with dexamethasone reversed this defect. During fasting, when the ATP-dependent process rises, muscles show a two- to threefold increase in levels of ubiquitin (Ub) mRNA. However, muscles of ADX animals failed to show this response. Injecting dexamethasone into the fasted ADX animals increased muscle Ub mRNA within 6 h. Thus glucocorticoids activate the ATP-Ub-dependent proteolytic pathway in fasting apparently by enhancing the expression of components of this system such as Ub.

  11. Longterm persistence of proteolytic activities in frass of Blattella germanica increases its allergenic potential.

    PubMed

    Erban, T; Hubert, J

    2011-06-01

    Chromogenic microplate assays in 96 wells were used to determine the stability of enzyme activity in frass of Blattella germanica (Blattodea: Blattellidae). Frass samples were exposed to controlled conditions [temperature 15-35 °C and/or 53-100% relative humidity (RH)] and to household conditions (apartment). Exposure times were 0 (control), 90, 183 and 276 days. Starch digestion and cellulolytic activities decreased during exposure. Non-specific proteolytic activities were affected by changes in selective proteolytic activities. Activities towards AAPpNA and SA(3) pNA strongly increased at 100% RH, indicating the possible influence of microorganisms growing on frass. Activities towards BApNA and ArgpNA decreased with increasing decomposition time, whereas activity towards ZRRpNA was not influenced by exposure time. The largest decrease in activities towards ArgpNA and BApNA occurred at temperatures of 15 °C, 30 °C and 35 °C and at 100% RH. Activities towards BApNA and ZRRpNA were very stable under different temperature and RH conditions; this was confirmed by findings showing that these activities were stable in the experimental apartment. In comparison with the control, activities towards ZRRpNA and BApNA after 276 days decreased by 1% and 19%, respectively. The longterm persistence of proteolytic activities in cockroach frass increases their allergenic hazard potential.

  12. PAI-1 (Plasminogen Activator Inhibitor-1) Expression Renders Alternatively Activated Human Macrophages Proteolytically Quiescent.

    PubMed

    Hohensinner, Philipp J; Baumgartner, Johanna; Kral-Pointner, Julia B; Uhrin, Pavel; Ebenbauer, Benjamin; Thaler, Barbara; Doberer, Konstantin; Stojkovic, Stefan; Demyanets, Svitlana; Fischer, Michael B; Huber, Kurt; Schabbauer, Gernot; Speidl, Walter S; Wojta, Johann

    2017-10-01

    Macrophages are versatile immune cells capable of polarizing into functional subsets depending on environmental stimulation. In atherosclerotic lesions, proinflammatory polarized macrophages are associated with symptomatic plaques, whereas Th2 (T-helper cell type 2) cytokine-polarized macrophages are inversely related with disease progression. To establish a functional cause for these observations, we analyzed extracellular matrix degradation phenotypes in polarized macrophages. We provide evidence that proinflammatory polarized macrophages rely on membrane-bound proteases including MMP-14 (matrix metalloproteinase-14) and the serine protease uPA (urokinase plasminogen activator) together with its receptor uPAR for extracellular matrix degradation. In contrast, Th2 cytokine alternatively primed macrophages do not show different proteolytic activity in comparison to unpolarized macrophages and lack increased localization of MMP-14 and uPA receptor to the cell membrane. Nonetheless, they express the highest amount of the serine protease uPA. However, uPA activity is blocked by similarly increased expression of its inhibitor PAI-1 (plasminogen activator inhibitor 1). When inhibiting PAI-1 or when analyzing macrophages deficient in PAI-1, Th2 cytokine-polarized macrophages display the same matrix degradation capability as proinflammatory-primed macrophages. Within atherosclerotic lesions, macrophages positive for the alternative activation marker CD206 express high levels of PAI-1. In addition, to test changed tissue remodeling capacities of alternatively activated macrophages, we used a bleomycin lung injury model in mice reconstituted with PAI-1(-/-) bone marrow. These results supported an enhanced remodeling phenotype displayed by increased fibrosis and elevated MMP activity in the lung after PAI-1 loss. We were able to demonstrate matrix degradation dependent on membrane-bound proteases in proinflammatory stimulated macrophages and a forced proteolytical quiescence

  13. Proteolytic, edematogenic and myotoxic activities of a hemorrhagic metalloproteinase isolated from Bothrops alternatus venom.

    PubMed

    Gay, C C; Leiva, L C; Maruñak, S; Teibler, P; Acosta de Pérez, O

    2005-10-01

    A hemorrhagic metalloproteinase has been isolated from Bothrops alternatus venom from specimens that inhabit the north-east region of Argentina. The present study aimed at evaluating the proteolytic, hemorrhagic, edematogenic and myotoxic activities of the purified metalloproteinase, in order to consider its participation on the phatophysiology of the intoxication by Bothrops alternatus venom. The hemorrhagic metalloproteinase was isolated by a combination of DEAE-Cellulose chromatography and gel filtration on Sephadex G-75. The enzyme showed a molecular mass around 55k Da, it exhibited a hemorrhagic activity with a minimal hemorrhagic dose of 1.9 microg, almost two fold minor than the whole venom (3.6 microg). The enzyme showed a weak proteolytic activity on casein (18.72 U/mg enzyme), similar to the one exhibited by the whole venom (20 U/mg venom). Besides, the ability to degrade casein could be detected by SDS-PAGE; beta-casein was the fraction that showed the higher degradation, followed by alphas(1)-casein and kappa-casein degradation. The hemorrhagic metalloproteinase rapidly hydrolysed the A alpha-chain of fibrinogen, followed by B beta-chain degradation and leaving the gamma-chain unaffected. Proteolytic activities were inhibited by EDTA whereas they were not inhibited by benzamidine and PMSF. The metalloproteinase showed several polypeptides chains after autocatalytic processing, including a chain of 28k Da, it could be the processed disintegrin-like and cysteine-rich domains. The isolated enzyme exhibited myotoxic activity with high CK levels at 6h, due to local ischemia resulting of its hemorrhagic activity, and a significant edema-inducing effect (MED=1.3 microg), corroborated both results by the histological observations of samples of gastrocnemius muscle. These findings showed that this hemorrhagic metalloproteinase, possesses high edematogenic and myotoxic activities and, in despite of exhibiting a weak proteolytic activity, it is able to degrade

  14. A new method for monitoring the extracellular proteolytic activity of wine yeasts during alcoholic fermentation of grape must.

    PubMed

    Chasseriaud, Laura; Miot-Sertier, Cécile; Coulon, Joana; Iturmendi, Nerea; Moine, Virginie; Albertin, Warren; Bely, Marina

    2015-12-01

    The existing methods for testing proteolytic activity are time consuming, quite difficult to perform, and do not allow real-time monitoring. Proteases have attracted considerable interest in winemaking and some yeast species naturally present in grape must, such as Metschnikowia pulcherrima, are capable of expressing this activity. In this study, a new test is proposed for measuring proteolytic activity directly in fermenting grape must, using azocasein, a chromogenic substrate. Several yeast strains were tested and differences in proteolytic activity were observed. Moreover, analysis of grape must proteins in wines revealed that protease secreted by Metschnikowia strains may be active against wine proteins.

  15. Influence of autoclaved saprotrophic fungal mycelia on proteolytic activity in ectomycorrhizal fungi.

    PubMed

    Mucha, Joanna; Dahm, Hanna; Werner, Antoni

    2007-07-01

    The production of proteolytic enzymes by several strains of ectomycorrhizal fungi i.e., Amanita muscaria (16-3), Laccaria laccata (9-12), L. laccata (9-1), Suillus bovinus (15-4), Suillus bovinus (15-3), Suillus luteus (14-7) on mycelia of Trichoderma harzianum, Trichoderma virens and Mucor hiemalis and sodium caseinate, yeast extract was evaluated. The strains of A. muscaria (16-3) and L. laccata (9-12) were characterized by the highest activity of the acidic and neutral proteases. Taking the mycelia of saprotrophic fungi into consideration, the mycelium of M. hiemalis was the best inductor for proteolytic activity. The examined ectomycorrhizal fungi exhibited higher activity of acidic proteases than neutral ones on the mycelia of saprotrophic fungi, which may imply the participation of acidic proteases in nutrition.

  16. Activation of bean (Phaseolus vulgaris) [alpha]-amylase inhibitor requires proteolytic processing of the proprotein

    SciTech Connect

    Pueyo, J.J.; Hunt, D.C.; Chrispeels, M.J. )

    1993-04-01

    Seeds of the common bean (Phaseolus vulgaris) contain a plant defense protein that inhibits the [alpha]-amylases of mammals and insects. This [alpha]-amylase inhibitor ([alpha]Al) is synthesized as a proprotein on the endoplasmic reticulum and is proteolytically processed after arrival in the protein storage vacuoles to polypeptides of relative molecular weight (M[sub r]) 15,000 to 18,000. The authors report two types of evidence that proteolytic processing is linked to activation of the inhibitory activity. First, by surveying seed extracts of wild accessions of P. vulgaris and other species in the genus Phaseolus, they found that antibodies to [alpha]Al recognize large (M[sub r] 30,000-35,000) polypeptides as well as typical [alpha]Al processing products (M[sub r] 15,000-18,000). [alpha]Al activity was found in all extracts that had the typical [alpha]Al processed polypeptides, but was absent from seed extracts that lacked such polypeptides. Second, they made a mutant [alpha]Al in which asparagine-77 is changed to aspartic acid-77. This mutation slows down the proteolytic processing of pro-[alpha]Al when the gene is expressed in tobacco. When pro-[alpha]Al was separated from mature [alpha]Al by gel filtration, pro-[alpha]Al was found not to have [alpha]-amylase inhibitory activity. The authors interpret these results to mean that formation of the active inhibitor is causally related to proteolytic processing of the proprotein. They suggest that the polypeptide cleavage removes a conformation constraint on the precursor to produce the biochemically active molecule. 43 refs., 5 figs., 1 tab.

  17. Ubiquitin, Proteasomes and Proteolytic Mechanisms Activated by Kidney Disease

    PubMed Central

    Rajan, Vik; Mitch, William E.

    2008-01-01

    Summary The ubiquitin-proteasome system (UPS) includes 3 enzymes that conjugate ubiquitin to intracellular proteins that are then recognized and degraded in the proteasome. The process participates in the regulation of cell metabolism. In the kidney, the UPS regulates the turnover of transporters and signaling proteins and its activity is down regulated in acidosis-induced proximal tubular cell hypertrophy. In chronic kidney disease (CKD), muscle wasting occurs because complications of CKD including acidosis, insulin resistance, inflammation, and increased angiotensin II levels stimulate the UPS to degrade muscle proteins. This response also includes caspase-3 and calpains which act to cleave muscle proteins to provide substrates for the UPS. For example, caspase-3 degrades actomyosin, leaving a 14kD fragment of actin in muscle. The 14 kD actin fragment is increased in muscle of patient with kidney disease, burn injury and surgery. In addition, acidosis, insulin resistance, inflammation and angiotensin II stimulate glucocorticoid production. Glucocorticoids are also required for the muscle wasting that occurs in CKD. Thus, the UPS is involved in regulating kidney function and participates in highly organized responses that degrade muscle protein in response to loss of kidney function. PMID:18723090

  18. Multifunctional Concentric FRET-Quantum Dot Probes for Tracking and Imaging of Proteolytic Activity.

    PubMed

    Massey, Melissa; Li, Jia Jun; Algar, W Russ

    2017-01-01

    Proteolysis has many important roles in physiological regulation. It is involved in numerous cell signaling processes and the pathogenesis of many diseases, including cancers. Methods of visualizing and assaying proteolytic activity are therefore in demand. Förster resonance energy transfer (FRET) probes offer several advantages in this respect. FRET supports end-point or real-time measurements, does not require washing or separation steps, and can be implemented in various assay or imaging formats. In this chapter, we describe methodology for preparing self-assembled concentric FRET (cFRET) probes for multiplexed tracking and imaging of proteolytic activity. The cFRET probe comprises a green-emitting semiconductor quantum dot (QD) conjugated with multiple copies of two different peptide substrates for two target proteases. The peptide substrates are labeled with different fluorescent dyes, Alexa Fluor 555 and Alexa Fluor 647, and FRET occurs between the QD and both dyes, as well as between the two dyes. This design enables a single QD probe to track the activity of two proteases simultaneously. Fundamental cFRET theory is presented, and procedures for using the cFRET probe for quantitative measurement of the activity of two model proteases are given, including calibration, fluorescence plate reader or microscope imaging assays, and data analysis. Sufficient detail is provided for other researchers to adapt this method to their specific requirements and proteolytic systems of interest.

  19. The Proteolytic Activity of Philibertia gilliesii Latex. Purification of Philibertain g II.

    PubMed

    Sequeiros, Cynthia; Torres, María J; Nievas, Marina L; Caffini, Néstor O; Natalucci, Claudia L; López, Laura M I; Trejo, Sebastián A

    2016-05-01

    The latex from the patagonic plant Philibertia gilliesii Hook. et Arn. (Apocynaceae) is a milky-white suspension containing a proteolytic system constituted by several cysteine endopeptidases. A proteolytic preparation (philibertain g) from the latex of P. gilliesii fruits was obtained and characterized to evaluate its potential use in bioprocesses. Philibertain g contained 1.2 g/L protein and a specific (caseinolytic) activity of 7.0 Ucas/mg protein. It reached 80 % of its maximum caseinolytic activity in the pH 7-10 range, retained 80 % of the original activity after 2 h of incubation at temperatures ranging from 25 to 45 °C and could be fully inactivated after 5 min at 75 °C. Philibertain g retained 60 % of the initial activity even at 1 M NaCl and was able to hydrolyze proteins from stickwater one, of the main waste effluents generated during fishmeal production. Furthermore, as a contribution to the knowledge of the proteolytic system of P. gilliesii, we are reporting the purification of a new peptidase, named philibertain g II (pI 9.4, molecular mass 23,977 Da, N-terminus LPESVDWREKGVVFPXRNQ) isolated from philibertain g through a purification scheme including acetone fractionation, cation exchange, molecular exclusion chromatography, and ultrafiltration.

  20. v-Src Oncogene Induces Trop2 Proteolytic Activation via Cyclin D1.

    PubMed

    Ju, Xiaoming; Jiao, Xuanmao; Ertel, Adam; Casimiro, Mathew C; Di Sante, Gabriele; Deng, Shengqiong; Li, Zhiping; Di Rocco, Agnese; Zhan, Tingting; Hawkins, Adam; Stoyanova, Tanya; Andò, Sebastiano; Fatatis, Alessandro; Lisanti, Michael P; Gomella, Leonard G; Languino, Lucia R; Pestell, Richard G

    2016-11-15

    Proteomic analysis of castration-resistant prostate cancer demonstrated the enrichment of Src tyrosine kinase activity in approximately 90% of patients. Src is known to induce cyclin D1, and a cyclin D1-regulated gene expression module predicts poor outcome in human prostate cancer. The tumor-associated calcium signal transducer 2 (TACSTD2/Trop2/M1S1) is enriched in the prostate, promoting prostate stem cell self-renewal upon proteolytic activation via a γ-secretase cleavage complex (PS1, PS2) and TACE (ADAM17), which releases the Trop2 intracellular domain (Trop2 ICD). Herein, v-Src transformation of primary murine prostate epithelial cells increased the proportion of prostate cancer stem cells as characterized by gene expression, epitope characteristics, and prostatosphere formation. Cyclin D1 was induced by v-Src, and Src kinase induction of Trop2 ICD nuclear accumulation required cyclin D1. Cyclin D1 induced abundance of the Trop2 proteolytic cleavage activation components (PS2, TACE) and restrained expression of the inhibitory component of the Trop2 proteolytic complex (Numb). Patients with prostate cancer with increased nuclear Trop2 ICD and cyclin D1, and reduced Numb, had reduced recurrence-free survival probability (HR = 4.35). Cyclin D1, therefore, serves as a transducer of v-Src-mediated induction of Trop2 ICD by enhancing abundance of the Trop2 proteolytic activation complex. Cancer Res; 76(22); 6723-34. ©2016 AACR. ©2016 American Association for Cancer Research.

  1. Unmasking Proteolytic Activity for Adult Visual Cortex Plasticity by the Removal of Lynx1.

    PubMed

    Bukhari, Noreen; Burman, Poromendro N; Hussein, Ayan; Demars, Michael P; Sadahiro, Masato; Brady, Daniel M; Tsirka, Stella E; Russo, Scott J; Morishita, Hirofumi

    2015-09-16

    Experience-dependent cortical plasticity declines with age. At the molecular level, experience-dependent proteolytic activity of tissue plasminogen activator (tPA) becomes restricted in the adult brain if mice are raised in standard cages. Understanding the mechanism for the loss of permissive proteolytic activity is therefore a key link for improving function in adult brains. Using the mouse primary visual cortex (V1) as a model, we demonstrate that tPA activity in V1 can be unmasked following 4 d of monocular deprivation when the mice older than 2 months are raised in standard cages by the genetic removal of Lynx1, a negative regulator of adult plasticity. This was also associated with the reduction of stubby and thin spine density and enhancement of ocular dominance shift in adult V1 of Lynx1 knock-out (KO) mice. These structural and functional changes were tPA-dependent because genetic removal of tPA in Lynx1 KO mice can block the monocular deprivation-dependent reduction of dendritic spine density, whereas both genetic and adult specific inhibition of tPA activity can ablate the ocular dominance shift in Lynx1 KO mice. Our work demonstrates that the adult brain has an intrinsic potential for experience-dependent elevation of proteolytic activity to express juvenile-like structural and functional changes but is effectively limited by Lynx1 if mice are raised in standard cages. Insights into the Lynx1-tPA plasticity mechanism may provide novel therapeutic targets for adult brain disorders. Experience-dependent proteolytic activity of tissue plasminogen activator (tPA) becomes restricted in the adult brain in correlation with the decline in cortical plasticity when mice are raised in standard cages. We demonstrated that removal of Lynx1, one of negative regulators of plasticity, unmasks experience-dependent tPA elevation in visual cortex of adult mice reared in standard cages. This proteolytic elevation facilitated dendritic spine reduction and ocular dominance

  2. Unmasking Proteolytic Activity for Adult Visual Cortex Plasticity by the Removal of Lynx1

    PubMed Central

    Bukhari, Noreen; Burman, Poromendro N.; Hussein, Ayan; Demars, Michael P.; Sadahiro, Masato; Brady, Daniel M.; Tsirka, Stella E.; Russo, Scott J.

    2015-01-01

    Experience-dependent cortical plasticity declines with age. At the molecular level, experience-dependent proteolytic activity of tissue plasminogen activator (tPA) becomes restricted in the adult brain if mice are raised in standard cages. Understanding the mechanism for the loss of permissive proteolytic activity is therefore a key link for improving function in adult brains. Using the mouse primary visual cortex (V1) as a model, we demonstrate that tPA activity in V1 can be unmasked following 4 d of monocular deprivation when the mice older than 2 months are raised in standard cages by the genetic removal of Lynx1, a negative regulator of adult plasticity. This was also associated with the reduction of stubby and thin spine density and enhancement of ocular dominance shift in adult V1 of Lynx1 knock-out (KO) mice. These structural and functional changes were tPA-dependent because genetic removal of tPA in Lynx1 KO mice can block the monocular deprivation-dependent reduction of dendritic spine density, whereas both genetic and adult specific inhibition of tPA activity can ablate the ocular dominance shift in Lynx1 KO mice. Our work demonstrates that the adult brain has an intrinsic potential for experience-dependent elevation of proteolytic activity to express juvenile-like structural and functional changes but is effectively limited by Lynx1 if mice are raised in standard cages. Insights into the Lynx1-tPA plasticity mechanism may provide novel therapeutic targets for adult brain disorders. SIGNIFICANCE STATEMENT Experience-dependent proteolytic activity of tissue plasminogen activator (tPA) becomes restricted in the adult brain in correlation with the decline in cortical plasticity when mice are raised in standard cages. We demonstrated that removal of Lynx1, one of negative regulators of plasticity, unmasks experience-dependent tPA elevation in visual cortex of adult mice reared in standard cages. This proteolytic elevation facilitated dendritic spine reduction

  3. Activation of the Aspergillus PacC zinc finger transcription factor requires two proteolytic steps

    PubMed Central

    Díez, Eliecer; Álvaro, Josué; Espeso, Eduardo A.; Rainbow, Lynne; Suárez, Teresa; Tilburn, Joan; Arst, Herbert N.; Peñalva, Miguel Á.

    2002-01-01

    The Aspergillus PacC transcription factor undergoes proteolytic activation in response to alkaline ambient pH. In acidic environments, the 674 residue translation product adopts a ‘closed’ conformation, protected from activation through intramolecular interactions involving the ≤150 residue C-terminal domain. pH signalling converts PacC to an accessible conformation enabling processing cleavage within residues 252–254. We demonstrate that activation of PacC requires two sequential proteolytic steps. First, the ‘closed’ translation product is converted to an accessible, committed intermediate by proteolytic elimination of the C-terminus. This ambient pH-regulated cleavage is required for the final, pH-independent processing reaction and is mediated by a distinct signalling protease (possibly PalB). The signalling protease cleaves PacC between residues 493 and 500, within a conserved 24 residue ‘signalling protease box’. Precise deletion or Leu498Ser substitution prevents formation of the committed and processed forms, demonstrating that signalling cleavage is essential for final processing. In contrast, signalling cleavage is not required for processing of the Leu340Ser protein, which lacks interactions preventing processing. In its two-step mechanism, PacC processing can be compared with regulated intramembrane proteolysis. PMID:11889040

  4. Proteolytic activity in Hysterothylacium aduncum (Nematoda: Anisakidae), a fish gastrointestinal parasite of worldwide distribution.

    PubMed

    Malagón, David; Benítez, Rocío; Adroher, Francisco Javier; Díaz-López, Manuel

    2011-12-29

    Proteases have a significant role in the life cycle of parasites and the pathogen-host relationship, being regarded as important virulence factors. In the parasitic nematode Hysterothylacium aduncum proteolytic activity was measured during in vitro development from third larval stage (L3) to mature adult, using DQ red casein as a fluorogenic substrate. Proteolytic activity was detected in all the developmental stages studied and at all pH values within the range employed (2.0-7.5). The assay with specific inhibitors permitted the determination of metalloprotease activity, and, to a lesser extent, that of aspartate- and cysteine-protease. Serine-protease activity was the lowest of those studied. In L3 recently collected from the host fish (L3-0 h), the greatest activity was found at an optimum pH of 4.0 and was mainly inhibited by 1,10-phenathroline (metalloprotease inhibitor). This metalloprotease activity in L3-0 h (infective stage) may be related to the invasion of the host tissues by this larva. In the other developmental stages, the greatest protease activity was found at pH 5.5, although at pH 4.0 a lower activity peak was detected. On the other hand, our data show that the proteolytic activity of the nematode varies according to the presence of pepsin (an aspartic-protease) in the culture medium. Thus, at pH 4.0, activity was greater in the absence of pepsin, with increasing aspartic-protease activity. Together with the detection of aspartic-, cysteine- and metallo-protease (enzymes involved in digestion in invertebrates) in all the developmental stages of the parasite taking place in the digestive tract of the host fish, this allows us to suggest that the pepsin in the culture medium mimics the predigestion conditions in the habitat of the worm within the host and that the activity detected may have, amongst others, a digestive function.

  5. Proteolytic activity monitored by fluorescence resonance energy transfer through quantum-dot-peptide conjugates

    NASA Astrophysics Data System (ADS)

    Medintz, Igor L.; Clapp, Aaron R.; Brunel, Florence M.; Tiefenbrunn, Theresa; Tetsuo Uyeda, H.; Chang, Eddie L.; Deschamps, Jeffrey R.; Dawson, Philip E.; Mattoussi, Hedi

    2006-07-01

    Proteases are enzymes that catalyse the breaking of specific peptide bonds in proteins and polypeptides. They are heavily involved in many normal biological processes as well as in diseases, including cancer, stroke and infection. In fact, proteolytic activity is sometimes used as a marker for some cancer types. Here we present luminescent quantum dot (QD) bioconjugates designed to detect proteolytic activity by fluorescence resonance energy transfer. To achieve this, we developed a modular peptide structure which allowed us to attach dye-labelled substrates for the proteases caspase-1, thrombin, collagenase and chymotrypsin to the QD surface. The fluorescence resonance energy transfer efficiency within these nanoassemblies is easily controlled, and proteolytic assays were carried out under both excess enzyme and excess substrate conditions. These assays provide quantitative data including enzymatic velocity, Michaelis-Menten kinetic parameters, and mechanisms of enzymatic inhibition. We also screened a number of inhibitory compounds against the QD-thrombin conjugate. This technology is not limited to sensing proteases, but may be amenable to monitoring other enzymatic modifications.

  6. Molecular mechanisms for the conversion of zymogens to active proteolytic enzymes.

    PubMed Central

    Khan, A. R.; James, M. N.

    1998-01-01

    Proteolytic enzymes are synthesized as inactive precursors, or "zymogens," to prevent unwanted protein degradation, and to enable spatial and temporal regulation of proteolytic activity. Upon sorting or appropriate compartmentalization, zymogen conversion to the active enzyme typically involves limited proteolysis and removal of an "activation segment." The sizes of activation segments range from dipeptide units to independently folding domains comprising more than 100 residues. A common form of the activation segment is an N-terminal extension of the mature enzyme, or "prosegment," that sterically blocks the active site, and thereby prevents binding of substrates. In addition to their inhibitory role, prosegments are frequently important for the folding, stability, and/or intracellular sorting of the zymogen. The mechanisms of conversion to active enzymes are diverse in nature, ranging from enzymatic or nonenzymatic cofactors that trigger activation, to a simple change in pH that results in conversion by an autocatalytic mechanism. Recent X-ray crystallographic studies of zymogens and comparisons with their active counterparts have identified the structural changes that accompany conversion. This review will focus upon the structural basis for inhibition by activation segments, as well as the molecular events that lead to the conversion of zymogens to active enzymes. PMID:9568890

  7. Different patterns of extracellular proteolytic activity in W303a and BY4742 Saccharomyces cerevisiae strains.

    PubMed

    Seredyński, Rafał; Wolna, Dorota; Kędzior, Mateusz; Gutowicz, Jan

    2017-01-01

    Protease secretion in Saccharomyces cerevisiae cultures is a complex process, important for the application of this organism in the food industry and biotechnology. Previous studies provide rather quantitative data, yielding no information about the number of enzymes involved in proteolysis and their individual biochemical properties. Here we demonstrate that W303a and BY4742 S. cerevisiae strains reveal different patterns of spontaneous and gelatin-induced extracellular proteolytic activity. We applied the gelatin zymography assay to track changes of the proteolytic profile in time, finding the protease secretion dependent on the growth phase and the presence of the protein inducer. Detected enzymes were characterized regarding their substrate specificity, pH tolerance, and susceptibility to inhibitors. In case of the W303a strain, only one type of gelatin-degrading secretory protease (presumably metalloproteinase) was observed. However, the BY4742 strain secreted different proteases of the various catalytic types, depending on the substrate availability. Our study brings the evidence that S. cerevisiae strains secrete several kinds of proteases depending on the presence and type of the substrate. Protein induction may cause not only quantitative but also qualitative changes in the extracellular proteolytic patterns.

  8. Measurement of Separase Proteolytic Activity in Single Living Cells by a Fluorogenic Flow Cytometry Assay

    PubMed Central

    Haaß, Wiltrud; Kleiner, Helga; Müller, Martin C.; Hofmann, Wolf-Karsten; Fabarius, Alice; Seifarth, Wolfgang

    2015-01-01

    ESPL1/Separase, an endopeptidase, is required for centrosome duplication and separation of sister-chromatides in anaphase of mitosis. Overexpression and deregulated proteolytic activity of Separase as frequently observed in human cancers is associated with the occurrence of supernumerary centrosomes, chromosomal missegregation and aneuploidy. Recently, we have hypothesized that increased Separase proteolytic activity in a small subpopulation of tumor cells may serve as driver of tumor heterogeneity and clonal evolution in chronic myeloid leukemia (CML). Currently, there is no quantitative assay to measure Separase activity levels in single cells. Therefore, we have designed a flow cytometry-based assay that utilizes a Cy5- and rhodamine 110 (Rh110)-biconjugated Rad21 cleavage site peptide ([Cy5-D-R-E-I-M-R]2-Rh110) as smart probe and intracellular substrate for detection of Separase enzyme activity in living cells. As measured by Cy5 fluorescence the cellular uptake of the fluorogenic peptide was fast and reached saturation after 210 min of incubation in human histiocytic lymphoma U937 cells. Separase activity was recorded as the intensity of Rh110 fluorescence released after intracellular peptide cleavage providing a linear signal gain within a 90–180 min time slot. Compared to conventional cell extract-based methods the flow cytometric assay delivers equivalent results but is more reliable, bypasses the problem of vague loading controls and unspecific proteolysis associated with whole cell extracts. Especially suited for the investigaton of blood- and bone marrow-derived hematopoietic cells the flow cytometric Separase assay allows generation of Separase activity profiles that tell about the number of Separase positive cells within a sample i.e. cells that currently progress through mitosis and about the range of intercellular variation in Separase activity levels within a cell population. The assay was used to quantify Separase proteolytic activity in leukemic

  9. VMY-1-103, a dansylated analog of purvalanol B, induces caspase-3-dependent apoptosis in LNCaP prostate cancer cells.

    PubMed

    Ringer, Lymor; Sirajuddin, Paul; Yenugonda, Venkata Mahidhar; Ghosh, Anup; Divito, Kyle; Trabosh, Valerie; Patel, Yesha; Brophy, Amanda; Grindrod, Scott; Lisanti, Michael P; Rosenthal, Dean; Brown, Milton L; Avantaggiati, Maria Laura; Rodriguez, Olga; Albanese, Chris

    2010-08-15

    The 2,6,9-trisubstituted purine group of cyclin dependent kinase inhibitors have the potential to be clinically relevant inhibitors of cancer cell proliferation. We have recently designed and synthesized a novel dansylated analog of purvalanol B, termed VMY-1-103, that inhibited cell cycle progression in breast cancer cell lines more effectively than did purvalanol B and allowed for uptake analyses by fluorescence microscopy. ErbB-2 plays an important role in the regulation of signal transduction cascades in a number of epithelial tumors, including prostate cancer (PCa). Our previous studies demonstrated that transgenic expression of activated ErbB-2 in the mouse prostate initiated PCa and either the overexpression of ErbB-2 or the addition of the ErbB-2/ErbB-3 ligand, heregulin (HRG), induced cell cycle progression in the androgen-responsive prostate cancer cell line, LNCaP. In the present study, we tested the efficacy of VMY-1-103 in inhibiting HRG-induced cell proliferation in LNCaP prostate cancer cells. At concentrations as low as 1 μM, VMY-1-103 increased both the proportion of cells in G(1) and p21(CIP1) protein levels. At higher concentrations (5 μM or 10 μM), VMY-1-103 induced apoptosis via decreased mitochondrial membrane polarity and induction of p53 phosphorylation, caspase-3 activity and PARP cleavage. Treatment with 10 μM Purvalanol B failed to either influence proliferation or induce apoptosis. Our results demonstrate that VMY-1-103 was more effective in inducing apoptosis in PCa cells than its parent compound, purvalanol B, and support the testing of VMY-1-103 as a potential small molecule inhibitor of prostate cancer in vivo.

  10. Neutrophil proteolytic activation cascades: a possible mechanistic link between chronic periodontitis and coronary heart disease.

    PubMed

    Alfakry, Hatem; Malle, Ernst; Koyani, Chintan N; Pussinen, Pirkko J; Sorsa, Timo

    2016-01-01

    Cardiovascular diseases are chronic inflammatory diseases that affect a large segment of society. Coronary heart disease (CHD), the most common cardiovascular disease, progresses over several years and affects millions of people worldwide. Chronic infections may contribute to the systemic inflammation and enhance the risk for CHD. Periodontitis is one of the most common chronic infections that affects up to 50% of the adult population. Under inflammatory conditions the activation of endogenous degradation pathways mediated by immune responses leads to the release of destructive cellular molecules from both resident and immigrant cells. Matrix metalloproteinases (MMPs) and their regulators can activate each other and play an important role in immune response via degrading extracellular matrix components and modulating cytokines and chemokines. The action of MMPs is required for immigrant cell recruitment at the site of inflammation. Stimulated neutrophils represent the major pathogen-fighting immune cells that upregulate expression of several proteinases and oxidative enzymes, which can degrade extracellular matrix components (e.g. MMP-8, MMP-9 and neutrophil elastase). The activity of MMPs is regulated by endogenous inhibitors and/or candidate MMPs (e.g. MMP-7). The balance between MMPs and their inhibitors is thought to mirror the proteolytic burden. Thus, neutrophil-derived biomarkers, including myeloperoxidase, may activate proteolytic destructive cascades that are involved in subsequent immune-pathological events associated with both periodontitis and CHD. Here, we review the existing studies on the contribution of MMPs and their regulators to the infection-related pathology. Also, we discuss the possible proteolytic involvement and role of neutrophil-derived enzymes as an etiological link between chronic periodontitis and CHD.

  11. Characterization of sodium dodecyl sulfate-resistant proteolytic activity in the hyperthermophilic archaebacterium Pyrococcus furiosus

    SciTech Connect

    Blumentals, I.I.; Robinson, A.S.; Kelly, R.M. )

    1990-07-01

    Cell extracts from Pyrococcus furiosus were found to contain five proteases, two of which (S66 and S102) are resistant to sodium dodecyl sulfate (SDS) denaturation. Cell extracts incubated at 98{degree}C in the presence of 1% SDS for 24 h exhibited substantial cellular proteolysis such that only four proteins could be visualized by amido black-Coomassie brilliant blue staining of SDS-polyacrylamide gels. The SDS-treated extract retained 19% of the initial proteolytic activity as represented by two proteases, S66 (66 kilodaltons (kDa)) and S102 (102 kDa). Immunoblot analysis with guinea pig sera containing antibodies against protease S66 indicated that S66 is related neither to S102 nor to the other proteases. The results of this analysis also suggest that S66 might be the hydrolysis product of a 200-kDa precursor which does not have proteolytic activity. The 24-h SDS-treated extract showed unusually thermostable proteolytic activity; the measured half-life at 98{degree}C was found to be 33 h. Proteases S66 and S102 were also resistant to denaturation by 8 M urea, 80 mM dithiothreitol, and 5% {beta}-mercaptoethanol. Purified protease S66 was inhibited by phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate but not by EDTA, ethylene glycol-bis({beta}-aminoethyl ether)-N,N,N{prime},N{prime}-tetraacetic acid, or iodoacetic acid. These results indicate that S66 is a serine protease. Amino acid ester hydrolysis studies showed that protease S66 was hydrolytically active towards N-benzoyl-L-arginine ethyl ester.

  12. Rock squirrel (Spermophilus variegatus) blood sera affects proteolytic and hemolytic activities of rattlesnake venoms.

    PubMed

    Biardi, James E; Coss, Richard G

    2011-02-01

    Rock squirrels (Spermophilus variegatus) from two sites in south central New Mexico, where prairie (Crotalus viridis viridis) and western diamondback (Crotalus atrox) rattlesnakes are common predators, were assayed for inhibition of rattlesnake venom digestive and hemostatic activities. At statistically significant levels rock squirrel blood sera reduced the metalloprotease and hemolytic activity of venoms from C. v. viridis and C. atrox more than venom from an allopatric snake species, the northern Pacific rattlesnake (Crotalus oreganus). In contrast, general proteolytic activity of venom from C. oreganus was inhibited more by S. variegatus serum defenses than activity of venom from sympatric snakes. For all three venoms, incubation with squirrel sera increased the level of fibrinolysis over venom-only treatments. These results suggest that rock squirrels (S. variegatus) can defend against metalloproteases and other proteases after envenomation from at least two of five rattlesnake predators they might encounter. However, there were statistically significant differences between general proteolytic activity and fibrinolytic activity of C. v. viridis and C. atrox venom, suggesting that rock squirrels might be differentially vulnerable to these two predators. The hypothesis that prey resistance influences snake venom evolution in a predator-prey arms race is given further support by the previously cryptic variation in venoms detected when assayed against prey defenses. Copyright © 2010 Elsevier Ltd. All rights reserved.

  13. Matriptase shedding is closely coupled with matriptase zymogen activation and requires de novo proteolytic cleavage likely involving its own activity

    PubMed Central

    Barndt, Robert; Gu, Yayun; Chen, Chien-Yu; Tseng, I-Chu; Su, Sheng-Fang; Wang, Jehng-Kang; Johnson, Michael D.

    2017-01-01

    The type 2 transmembrane serine protease matriptase is involved in many pathophysiological processes probably via its enzymatic activity, which depends on the dynamic relationship between zymogen activation and protease inhibition. Matriptase shedding can prolong the life of enzymatically active matriptase and increase accessibility to substrates. We show here that matriptase shedding occurs via a de novo proteolytic cleavage at sites located between the SEA domain and the CUB domain. Point or combined mutations at the four positively charged amino acid residues in the region following the SEA domain allowed Arg-186 to be identified as the primary cleavage site responsible for matriptase shedding. Kinetic studies further demonstrate that matriptase shedding is temporally coupled with matriptase zymogen activation. The onset of matriptase shedding lags one minute behind matriptase zymogen activation. Studies with active site triad Ser-805 point mutated matriptase, which no longer undergoes zymogen activation or shedding, further suggests that matriptase shedding depends on matriptase zymogen activation, and that matriptase proteolytic activity may be involved in its own shedding. Our studies uncover an autonomous mechanism coupling matriptase zymogen activation, proteolytic activity, and shedding such that a proportion of newly generated active matriptase escapes HAI-1-mediated rapid inhibition by shedding into the extracellular milieu. PMID:28829816

  14. Biochemical and Functional Characterization of Parawixia bistriata Spider Venom with Potential Proteolytic and Larvicidal Activities

    PubMed Central

    Gimenez, Gizeli S.; Coutinho-Neto, Antonio; Kayano, Anderson M.; Simões-Silva, Rodrigo; Trindade, Frances; de Almeida e Silva, Alexandre; Marcussi, Silvana; da Silva, Saulo L.; Fernandes, Carla F. C.; Zuliani, Juliana P.; Calderon, Leonardo A.; Soares, Andreimar M.; Stábeli, Rodrigo G.

    2014-01-01

    Toxins purified from the venom of spiders have high potential to be studied pharmacologically and biochemically. These biomolecules may have biotechnological and therapeutic applications. This study aimed to evaluate the protein content of Parawixia bistriata venom and functionally characterize its proteins that have potential for biotechnological applications. The crude venom showed no phospholipase, hemorrhagic, or anti-Leishmania activities attesting to low genotoxicity and discrete antifungal activity for C. albicans. However the following activities were observed: anticoagulation, edema, myotoxicity and proteolysis on casein, azo-collagen, and fibrinogen. The chromatographic and electrophoretic profiles of the proteins revealed a predominance of acidic, neutral, and polar proteins, highlighting the presence of proteins with high molecular masses. Five fractions were collected using cation exchange chromatography, with the P4 fraction standing out as that of the highest purity. All fractions showed proteolytic activity. The crude venom and fractions P1, P2, and P3 showed larvicidal effects on A. aegypti. Fraction P4 showed the presence of a possible metalloprotease (60 kDa) that has high proteolytic activity on azo-collagen and was inhibited by EDTA. The results presented in this study demonstrate the presence of proteins in the venom of P. bistriata with potential for biotechnological applications. PMID:24895632

  15. Biochemical and functional characterization of Parawixia bistriata spider venom with potential proteolytic and larvicidal activities.

    PubMed

    Gimenez, Gizeli S; Coutinho-Neto, Antonio; Kayano, Anderson M; Simões-Silva, Rodrigo; Trindade, Frances; de Almeida e Silva, Alexandre; Marcussi, Silvana; da Silva, Saulo L; Fernandes, Carla F C; Zuliani, Juliana P; Calderon, Leonardo A; Soares, Andreimar M; Stábeli, Rodrigo G

    2014-01-01

    Toxins purified from the venom of spiders have high potential to be studied pharmacologically and biochemically. These biomolecules may have biotechnological and therapeutic applications. This study aimed to evaluate the protein content of Parawixia bistriata venom and functionally characterize its proteins that have potential for biotechnological applications. The crude venom showed no phospholipase, hemorrhagic, or anti-Leishmania activities attesting to low genotoxicity and discrete antifungal activity for C. albicans. However the following activities were observed: anticoagulation, edema, myotoxicity and proteolysis on casein, azo-collagen, and fibrinogen. The chromatographic and electrophoretic profiles of the proteins revealed a predominance of acidic, neutral, and polar proteins, highlighting the presence of proteins with high molecular masses. Five fractions were collected using cation exchange chromatography, with the P4 fraction standing out as that of the highest purity. All fractions showed proteolytic activity. The crude venom and fractions P1, P2, and P3 showed larvicidal effects on A. aegypti. Fraction P4 showed the presence of a possible metalloprotease (60 kDa) that has high proteolytic activity on azo-collagen and was inhibited by EDTA. The results presented in this study demonstrate the presence of proteins in the venom of P. bistriata with potential for biotechnological applications.

  16. Comparison of proteolytic activity of Candida sp. strains depending on their origin.

    PubMed

    Modrzewska, B; Kurnatowski, P; Khalid, K

    2016-06-01

    The aim of the research was to evaluate the proteolytic activity of various Candida strains isolated from the oral cavity of persons without clinical symptoms of fungal infection, outpatients with oral cavity disorders and patients hospitalized due to head and neck tumors. A secondary aim was to confirm the presence of secreted aspartyl protease (SAP) genes in the isolated strains and then to compare it depending on the fungal species. Material consisted of 134 fungal strains that were analysed by a modified Staib method and polymerase chain reaction (PCR) with the use of specific primer pairs. The greatest proteolytic activity of fungi was observed at pH 3.5. The proteolysis were the strongest for strains isolated from dental patients and the weakest from persons without changes in the oral cavity. In total, 61.9% of the strains exhibited the presence of at least one of the SAP1-3 genes in all examined groups, SAP1 being the most common; SAP4-6 genes were not observed. All genes were more frequent in the strains isolated from the dental patients than from other groups. SAP1-3 genes were present in Candida albicans, C. tropicalis, C. parapsilosis, C. glabrata, C. humicola and C. lipolytica, but were not noted in other isolated species. The lowest activity of proteolytic enzymes and the least number of aspartyl protease genes are observed among strains isolated from patients without clinical symptoms of mycosis. SAP1-3 genes are most frequently detected in the strains isolated from the oral cavity; their presence varies depending on the species of the fungi. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  17. Proteolytic properties of single-chain factor XII: a mechanism for triggering contact activation.

    PubMed

    Ivanov, Ivan; Matafonov, Anton; Sun, Mao-Fu; Cheng, Qiufang; Dickeson, S Kent; Verhamme, Ingrid M; Emsley, Jonas; Gailani, David

    2017-03-16

    When blood is exposed to variety of artificial surfaces and biologic substances, the plasma proteins factor XII (FXII) and prekallikrein undergo reciprocal proteolytic conversion to the proteases αFXIIa and α-kallikrein by a process called contact activation. These enzymes contribute to host-defense responses including coagulation, inflammation, and fibrinolysis. The initiating event in contact activation is debated. To test the hypothesis that single-chain FXII expresses activity that could initiate contact activation, we prepared human FXII variants lacking the Arg353 cleavage site required for conversion to αFXIIa (FXII-R353A), or lacking the 3 known cleavage sites at Arg334, Arg343, and Arg353 (FXII-T, for "triple" mutant), and compared their properties to wild-type αFXIIa. In the absence of a surface, FXII-R353A and FXII-T activate prekallikrein and cleave the tripeptide S-2302, demonstrating proteolytic activity. The activity is several orders of magnitude weaker than that of αFXIIa. Polyphosphate, an inducer of contact activation, enhances PK activation by FXII-T, and facilitates FXII-T activation of FXII and FXI. In plasma, FXII-T and FXII-R353A, but not FXII lacking the active site serine residue (FXII-S544A), shortened the clotting time of FXII-deficient plasma and enhanced thrombin generation in a surface-dependent manner. The effect was not as strong as for wild-type FXII. Our results support a model for induction of contact activation in which activity intrinsic to single-chain FXII initiates αFXIIa and α-kallikrein formation on a surface. αFXIIa, with support from α-kallikrein, subsequently accelerates contact activation and is responsible for the full procoagulant activity of FXII.

  18. Bromophenol blue protein assay: improvement in buffer tolerance and adaptation for the measurement of proteolytic activity.

    PubMed

    Ahmad, H; Saleemuddin, M

    1983-07-01

    A modification of the bromophenol blue dye binding procedure of protein estimation is described. Substitution of glycine/phosphoric acid, pH 2.6, for dilute acetic acid in the colour reagent extended the applicability of the procedure to protein solutions containing buffers of various pH values. This was, however, accompanied by approximately 25% loss in the sensitivity of the procedure. The modified reagent exhibited very marked tolerance to detergents and could be successfully adapted for the measurement of proteolytic activity in acidic, neutral or alkaline pH ranges.

  19. Purification and Properties of Two Proteolytic Enzymes with Carboxypeptidase Activity in Germinated Wheat 1

    PubMed Central

    Preston, Ken R.; Kruger, James E.

    1976-01-01

    Two proteolytic enzymes with carboxypeptidase activity have been isolated from a germinated wheat extract and partially characterized. Both enzymes rapidly released amino acids from hemoglobin and gluten and hydrolyzed carbobenzoxy-phenylalanylalanine. The enzymes were inhibited by diisopropylphosphofluoridate, but unaffected by salts, ethylenediaminetetraacetate, and sulfhydryl reagents at lower concentrations, and had molecular weights of approximately 55,000 and 61,000. Analysis of the hydrolysis products of hemoglobin and gluten indicated that both enzymes had broad specificities, including the ability to release proline. Images PMID:16659708

  20. Reconstitution of active catalytic trimer of aspartate transcarbamoylase from proteolytically cleaved polypeptide chains.

    PubMed Central

    Powers, V. M.; Yang, Y. R.; Fogli, M. J.; Schachman, H. K.

    1993-01-01

    Treatment of the catalytic (C) trimer of Escherichia coli aspartate transcarbamoylase (ATCase) with alpha-chymotrypsin by a procedure similar to that used by Chan and Enns (1978, Can. J. Biochem. 56, 654-658) has been shown to yield an intact, active, proteolytically cleaved trimer containing polypeptide fragments of 26,000 and 8,000 MW. Vmax of the proteolytically cleaved trimer (CPC) is 75% that of the wild-type C trimer, whereas Km for aspartate and Kd for the bisubstrate analog, N-(phosphonacetyl)-L-aspartate, are increased about 7- and 15-fold, respectively. CPC trimer is very stable to heat denaturation as shown by differential scanning microcalorimetry. Amino-terminal sequence analyses as well as results from electrospray ionization mass spectrometry indicate that the limited chymotryptic digestion involves the rupture of only a single peptide bond leading to the production of two fragments corresponding to residues 1-240 and 241-310. This cleavage site involving the bond between Tyr 240 and Ala 241 is in a surface loop known to be involved in intersubunit contacts between the upper and lower C trimers in ATCase when it is in the T conformation. Reconstituted holoenzyme comprising two CPC trimers and three wild-type regulatory (R) dimers was shown by enzyme assays to be devoid of the homotropic and heterotropic allosteric properties characteristic of wild-type ATCase. Moreover, sedimentation velocity experiments demonstrate that the holoenzyme reconstituted from CPC trimers is in the R conformation. These results indicate that the intact flexible loop containing Tyr 240 is essential for stabilizing the T conformation of ATCase. Following denaturation of the CPC trimer in 4.7 M urea and dilution of the solution, the separate proteolytic fragments re-associate to form active trimers in about 60% yield. How this refolding of the fragments, docking, and association to form trimers are achieved is not known. PMID:8318885

  1. Antifungal and proteolytic activities of endophytic fungi isolated from Piper hispidum Sw.

    PubMed

    Orlandelli, Ravely Casarotti; de Almeida, Tiago Tognolli; Alberto, Raiani Nascimento; Polonio, Julio Cesar; Azevedo, João Lúcio; Pamphile, João Alencar

    2015-06-01

    Endophytes are being considered for use in biological control, and the enzymes they secrete might facilitate their initial colonization of internal plant tissues and direct interactions with microbial pathogens. Microbial proteases are also biotechnologically important products employed in bioremediation processes, cosmetics, and the pharmaceutical, photographic and food industries. In the present study, we evaluated antagonism and competitive interactions between 98 fungal endophytes and Alternaria alternata, Colletotrichum sp., Phyllosticta citricarpa and Moniliophthora perniciosa. We also examined the proteolytic activities of endophytes grown in liquid medium and conducted cup plate assays. The results showed that certain strains in the assemblage of P. hispidum endophytes are important sources of antifungal properties, primarily Lasiodiplodia theobromae JF766989, which reduced phytopathogen growth by approximately 54 to 65%. We detected 28 endophytes producing enzymatic halos of up to 16.40 mm in diameter. The results obtained in the present study highlight the proteolytic activity of the endophytes Phoma herbarum JF766995 and Schizophyllum commune JF766994, which presented the highest enzymatic halo diameters under at least one culture condition tested. The increased activities of certain isolates in the presence of rice or soy flour as a substrate (with halos up to 17.67 mm in diameter) suggests that these endophytes have the potential to produce enzymes using agricultural wastes.

  2. Proteolytic activation of receptor-bound anthrax protective antigen on macrophages promotes its internalization.

    PubMed

    Beauregard, K E; Collier, R J; Swanson, J A

    2000-06-01

    Immunofluorescence and other methods have been used to probe the self-assembly and internalization of the binary toxin, anthrax lethal toxin (LeTx), in primary murine macrophages. Proteolytic activation of protective antigen (PA; 83 kDa, the B moiety of the toxin) by furin was the rate-limiting step in internalization of LeTx and promoted clearance of PA from the cell surface. A furin-resistant form of PA remained at the cell surface for at least 90 min. Oligomerization of receptor-bound PA63, the 63 kDa active fragment of PA, was manifested by its conversion to a pronase-resistant state, characteristic of the heptameric prepore form in solution. That oligomerization of PA63 triggers toxin internalization is supported by the observation that PA20, the complementary 20 kDa fragment of PA, inhibited clearance of nicked PA. The PA63 prepore, with or without lethal factor (LF), cleared slowly from the cell surface. These studies show that proteolytic cleavage of PA, in addition to permitting oligomerization and LF binding, also promotes internalization of the protein. The relatively long period of activation and internalization of PA at the cell surface may reflect adaptation of this binary toxin that maximizes self-assembly.

  3. Antifungal and proteolytic activities of endophytic fungi isolated from Piper hispidum Sw

    PubMed Central

    Orlandelli, Ravely Casarotti; de Almeida, Tiago Tognolli; Alberto, Raiani Nascimento; Polonio, Julio Cesar; Azevedo, João Lúcio; Pamphile, João Alencar

    2015-01-01

    Endophytes are being considered for use in biological control, and the enzymes they secrete might facilitate their initial colonization of internal plant tissues and direct interactions with microbial pathogens. Microbial proteases are also biotechnologically important products employed in bioremediation processes, cosmetics, and the pharmaceutical, photographic and food industries. In the present study, we evaluated antagonism and competitive interactions between 98 fungal endophytes and Alternaria alternata, Colletotrichum sp., Phyllosticta citricarpa and Moniliophthora perniciosa. We also examined the proteolytic activities of endophytes grown in liquid medium and conducted cup plate assays. The results showed that certain strains in the assemblage of P. hispidum endophytes are important sources of antifungal properties, primarily Lasiodiplodia theobromae JF766989, which reduced phytopathogen growth by approximately 54 to 65%. We detected 28 endophytes producing enzymatic halos of up to 16.40 mm in diameter. The results obtained in the present study highlight the proteolytic activity of the endophytes Phoma herbarum JF766995 and Schizophyllum commune JF766994, which presented the highest enzymatic halo diameters under at least one culture condition tested. The increased activities of certain isolates in the presence of rice or soy flour as a substrate (with halos up to 17.67 mm in diameter) suggests that these endophytes have the potential to produce enzymes using agricultural wastes. PMID:26273250

  4. Recovery of proteolytic and collagenolytic activities from viscera by-products of rayfish (Raja clavata).

    PubMed

    Murado, Miguel Anxo; González, María del Pilar; Vázquez, José Antonio

    2009-12-15

    The aim of this work was to study the recovery of proteolytic and collagenolytic activities from rayfish (Raja clavata) viscera wastes. Initially, different parts of the gastrointestinal tract by-products (stomach, duodenum section including pancreas, final intestine) were evaluated. The extracts from proximal intestine yielded the highest values of both enzymatic activities. Optimal conditions for protease activity quantification were established at pH = 6, T = 40 degrees C and incubation time < or =20 min. The mathematical equation used to model the joint effect of pH and temperature led to maximum activity at pH = 8.66 and 59.4 degrees C, respectively. Overcooled acetone was found to be best option for recovery of enzymatic activities in comparison with ethanol, PEG-4000, ammonium sulphate and ultrafiltration system. Finally, a simple and systematic protocol of partial purification and total recovery of proteases and collagenases was defined.

  5. Recovery of Proteolytic and Collagenolytic Activities from Viscera By-products of Rayfish (Raja clavata)

    PubMed Central

    Murado, Miguel Anxo; del Pilar González, María; Vázquez, José Antonio

    2009-01-01

    The aim of this work was to study the recovery of proteolytic and collagenolytic activities from rayfish (Raja clavata) viscera wastes. Initially, different parts of the gastrointestinal tract by-products (stomach, duodenum section including pancreas, final intestine) were evaluated. The extracts from proximal intestine yielded the highest values of both enzymatic activities. Optimal conditions for protease activity quantification were established at pH = 6, T = 40 °C and incubation time ≤20 min. The mathematical equation used to model the joint effect of pH and temperature led to maximum activity at pH = 8.66 and 59.4 °C, respectively. Overcooled acetone was found to be best option for recovery of enzymatic activities in comparison with ethanol, PEG-4000, ammonium sulphate and ultrafiltration system. Finally, a simple and systematic protocol of partial purification and total recovery of proteases and collagenases was defined. PMID:20098611

  6. Proteolytic activity of Enterococcus faecalis VB63F for reduction of allergenicity of bovine milk proteins.

    PubMed

    Biscola, V; Tulini, F L; Choiset, Y; Rabesona, H; Ivanova, I; Chobert, J-M; Todorov, S D; Haertlé, T; Franco, B D G M

    2016-07-01

    With the aim of screening proteolytic strains of lactic acid bacteria to evaluate their potential for the reduction of allergenicity of the major bovine milk proteins, we isolated a new proteolytic strain of Enterococcus faecalis (Ent. faecalis VB63F) from raw bovine milk. The proteases produced by this strain had strong activity against caseins (αS1-, αS2-, and β-casein), in both skim milk and sodium caseinate. However, only partial hydrolysis of whey proteins was observed. Proteolysis of Na-caseinate and whey proteins, observed after sodium dodecyl sulfate-PAGE, was confirmed by analysis of peptide profiles by reversed-phase HPLC. Inhibition of proteolysis with EDTA indicated that the proteases produced by Ent. faecalis VB63F belonged to the group of metalloproteases. The optimal conditions for their activity were 42°C and pH 6.5. The majority of assessed virulence genes were absent in Ent. faecalis VB63F. The obtained results suggest that Ent. faecalis VB63F could be efficient in reducing the immunoreactivity of bovine milk proteins.

  7. Soybean P34 Probable Thiol Protease Probably Has Proteolytic Activity on Oleosins.

    PubMed

    Zhao, Luping; Kong, Xiangzhen; Zhang, Caimeng; Hua, Yufei; Chen, Yeming

    2017-07-19

    P34 probable thiol protease (P34) and Gly m Bd 30K (30K) show high relationship with the protease of 24 kDa oleosin of soybean oil bodies. In this study, 9 day germinated soybean was used to separate bioprocessed P34 (P32) from bioprocessed 30K (28K). Interestingly, P32 existed as dimer, whereas 28K existed as monomer; a P32-rich sample had proteolytic activity and high cleavage site specificity (Lys-Thr of 24 kDa oleosin), whereas a 28K-rich sample showed low proteolytic activity; the P32-rich sample contained one thiol protease. After mixing with purified oil bodies, all P32 dimers were dissociated and bound to 24 kDa oleosins to form P32-24 kDa oleosin complexes. By incubation, 24 kDa oleosin was preferentially hydrolyzed, and two hydrolyzed products (HPs; 17 and 7 kDa) were confirmed. After most of 24 kDa oleosin was hydrolyzed, some P32 existed as dimer, and the other as P32-17 kDa HP. It was suggested that P32 was the protease.

  8. Proteolytic activity of Penicillium chrysogenum and Debaryomyces hansenii during controlled ripening of pork loins.

    PubMed

    Martín, Alberto; Asensio, Miguel A; Bermúdez, María E; Córdoba, María G; Aranda, Emilio; Córdoba, Juan J

    2002-09-01

    The role of micro-organisms on the ripening process of dry-cured ham, particularly with respect to proteolysis, is not clear. This is partially due to the lack of an adequate system to study changes on a sterile control meat product for long ripening times. Using a meat system based on sterile pork loins ripened under aseptic conditions for 106 days, the contribution to the proteolysis of two micro-organisms isolated from dry-cured ham has been established. Changes were studied by SDS-PAGE of sarcoplasmic and myofibrillar proteins, capillary zone electrophoresis (CZE) of low ionic strength-soluble nitrogen compounds, and HPLC of free amino acids. Debaryomyces hansenii Dh345 did not show any significant proteolytic activity. However, Penicillium chrysogenum Pg222 showed high proteolytic activity on myofibrillar proteins resulting in an increase in soluble nitrogen compounds. For this, P. chrysogenum Pg222 should be considered to be used as starter culture in meat products made using long ripening times.

  9. Self-assembled gold nanoparticle molecular probes for detecting proteolytic activity in vivo

    PubMed Central

    Mu, C. Jenny; LaVan, David A.; Langer, Robert S.; Zetter, Bruce R.

    2010-01-01

    Target-activatable fluorogenic probes based on gold nanoparticles (AuNPs) functionalized with self-assembled heterogeneous monolayers of dye-labeled peptides and poly(ethylene glycol) have been developed to visualize proteolytic activity in vivo. A one-step synthesis strategy that allows simple generation of surface defined AuNP probe libraries is presented as a means of tailoring and evaluating probe characteristics for maximal fluorescence enhancement after protease activation. Optimal AuNP probes targeted to trypsin and urokinase-type plasminogen activator required the incorporation of a dark quencher to achieve 5 to 8-fold signal amplification. These probes exhibited extended circulation time in vivo and high image contrast in a mouse tumor model. PMID:20146506

  10. Mechanical Allostery: Evidence for a Force Requirement in the Proteolytic Activation of Notch

    PubMed Central

    Gordon, Wendy R.; Zimmerman, Brandon; He, Li; Miles, Laura J.; Huang, Jiuhong; Tiyanont, Kittichoat; McArthur, Debbie G.; Aster, Jon C.; Perrimon, Norbert; Loparo, Joseph J.; Blacklow, Stephen C.

    2015-01-01

    Summary Ligands stimulate Notch receptors by inducing regulated intramembrane proteolysis (RIP) to produce a transcriptional effector. Notch activation requires unmasking of a metalloprotease cleavage site remote from the site of ligand binding, raising the question of how proteolytic sensitivity is achieved. Here, we show that application of physiologically relevant forces to the regulatory switch results in sensitivity to metalloprotease cleavage, and that bound ligands induce Notch signal transduction in cells only in the presence of applied mechanical force. Synthetic receptor-ligand systems that remove the native ligand-receptor interaction also activate Notch by inducing proteolysis of the regulatory switch. Together, these studies show that mechanical force exerted by signal-sending cells is required for ligand-induced Notch activation, and establish that force-induced proteolysis can act as a mechanism of cellular mechanotransduction. PMID:26051539

  11. Proteolytic activity and fatal gram-negative sepsis in burned mice: effect of exogenous proteinase inhibition.

    PubMed Central

    Neely, A N; Miller, R G; Holder, I A

    1994-01-01

    Circulating proteolytic activity (PA) increases following burn or surgical trauma. Challenging traumatized mice with the yeast Candida albicans further increases PA. Once a PA threshold has been passed, mortality increases as PA increases. The purposes of this study were to determine (i) if gram-negative bacterial challenge affects circulating PA and mortality as Candida challenge does and (ii) if proteinase inhibitor treatment with aprotinin, antithrombin III, and alpha 1-proteinase inhibitor decreases circulating PA and increases the survival of burned mice infected with a bacterium. For all bacteria tested (Proteus mirabilis, Pseudomonas aeruginosa, and Klebsiella pneumoniae), burn plus challenge significantly elevated PA and mortality above levels in mice that were only burned or only challenged. Quantitative culture counts indicated that the mice died of sepsis. Proteinase inhibitor treatment of mice burned and challenged with K. pneumoniae significantly decreased circulating PA, decreased the hepatic microbial load, and increased survival. Hence, in traumatized mice challenged with either C. albicans or gram-negative bacteria, a relationship exists between proteolytic load and subsequent septic death. Parallels between these animal studies and human studies are discussed. PMID:8188336

  12. Inhibition of intrinsic proteolytic activities moderates preanalytical variability and instability of human plasma.

    PubMed

    Yi, Jizu; Kim, Changki; Gelfand, Craig A

    2007-05-01

    Human plasma and serum proteins are subject to intrinsic proteolytic degradation both during and after blood collection. By monitoring peptides, we investigated the stability of plasma and serum samples and the effects of anticoagulants and protease inhibitors on the plasma samples. Serum and plasma were subjected to time-course incubation, and the peptides (750-3200 Da) were extracted and analyzed with MALDI-TOF MS. Peptides of interest were further identified by MALDI-TOF/TOF MS and ESI-MS/MS analyses. Our observations indicate that plasma peptides are significantly different from serum peptides. Intrinsic proteases cause these differences between plasma and serum samples, as well as the differences among three plasma samples using either EDTA, sodium citrate, or heparin as the anticoagulant, which accounts for partial inhibitory effects on plasma proteolytic activities. Proteases and peptidases, including both aminopeptidases and carboxypeptidases, also cause time-dependent, sequential generation and digestion of the peptides in serum and all three plasmas, specifically during early sample collection and processing. Protease inhibitors within an EDTA-plasma-collection device inhibit both intrinsic plasma peptidases and proteases and moderate the time-dependent changes of the plasma peptides, including bradykinin, and complement C4- and C3- derived peptides. Our results suggest that mixing protease inhibitors immediately with blood during blood collection provides enhanced stabilization of the plasma proteome.

  13. On how a transcription factor can avoid its proteolytic activation in the absence of signal transduction

    PubMed Central

    Espeso, Eduardo A.; Roncal, Tomás; Díez, Eliecer; Rainbow, Lynne; Bignell, Elaine; Álvaro, Josué; Suárez, Teresa; Denison, Steven H.; Tilburn, Joan; Arst, Herbert N.; Peñalva, Miguel A.

    2000-01-01

    In response to alkaline ambient pH, the Aspergillus nidulans PacC transcription factor mediating pH regulation of gene expression is activated by proteolytic removal of a negative-acting C–terminal domain. We demonstrate interactions involving the ∼150 C–terminal PacC residues and two regions located immediately downstream of the DNA binding domain. Our data indicate two full-length PacC conformations whose relative amounts depend upon ambient pH: one ‘open’ and accessible for processing, the other ‘closed’ and inaccessible. The location of essential determinants for proteolytic processing within the two more upstream interacting regions probably explains why the interactions prevent processing, whereas the direct involvement of the C–terminal region in processing-preventing interactions explains why C–terminal truncating mutations result in alkalinity mimicry and pH-independent processing. A mutant PacC deficient in pH signal response and consequent processing behaves as though locked in the ‘closed’ form. Single-residue substitutions, obtained as mutations bypassing the need for pH signal transduction, identify crucial residues in each of the three interactive regions and overcome the processing deficiency in the ‘permanently closed’ mutant. PMID:10675341

  14. Characterization of the proteolytic activity of starter cultures of Penicillium roqueforti for production of blue veined cheeses.

    PubMed

    Larsen, M D; Kristiansen, K R; Hansen, T K

    1998-09-08

    Thirty strains of Penicillium roqueforti used for the production of blue cheeses were examined for proteolytic activity by agar diffusion on casein agar, by the azocasein test and by capillary zone electrophoresis (CE). Distinct differences were seen between the strains by all three methods applied and the 30 strains could be subdivided in three groups being significantly different in their proteolytic activity as measured by the agar diffusion test. The quantitative differences seen in the agar diffusion test were confirmed by the azocasein test. However, a negative result on casein agar, i.e., no clearing of the agar was observed for one strain while it showed low proteolytic activity in the azocasein test. CE proved to be a valuable method for revealing qualitative differences between strains of P. roqueforti in the breakdown of casein. Three strongly proteolytic strains broke down the specific casein fractions differently: one strain broke down betaA1-casein faster than betaA2-casein, the second preferred alpha s1-casein while the last strain broke down the casein fractions at equal rates. For a strain with medium proteolytic activity, the degradation of casein was seen by the appearance of a peak with migration time similar to alpha s1-I casein, a peptide normally produced by chymosin.

  15. Activities of indigenous proteolytic enzymes in caprine milk of different somatic cell counts.

    PubMed

    Albenzio, M; Santillo, A; Kelly, A L; Caroprese, M; Marino, R; Sevi, A

    2015-11-01

    Individual caprine milk with different somatic cell counts (SCC) were studied with the aim of investigating the percentage distribution of leukocyte cell types and the activities of indigenous proteolytic enzymes; proteolysis of casein was also studied in relation to cell type following recovery from milk. The experiment was conducted on 5 intensively managed dairy flocks of Garganica goats; on the basis of SCC, the experimental groups were denoted low (L-SCC; <700,000 cells/mL), medium (M-SCC; from 701,000 to 1,500,000 cells/mL), and high (H-SCC; >1,501,000 cells/mL) SCC. Leukocyte distribution differed between groups; polymorphonuclear neutrophilic leukocytes were higher in M-SCC and H-SCC milk samples, the percentage macrophages was the highest in H-SCC, and levels of nonviable cells significantly decreased with increasing SCC. Activities of all the main proteolytic enzymes were affected by SCC; plasmin activity was the highest in H-SCC milk and the lowest in L-SCC, and elastase and cathepsin D activities were the highest in M-SCC. Somatic cell count influenced casein hydrolysis patterns, with less intact α- and β-casein in H-SCC milk. Higher levels of low electrophoretic mobility peptides were detected in sodium caseinate incubated with leukocytes isolated from L-SCC milk, independent of cell type, whereas among cells recovered from M-SCC milk, macrophages yielded the highest levels of low electrophoretic mobility peptides from sodium caseinate. The level of high electrophoretic mobility peptides was higher in sodium caseinate incubated with polymorphonuclear neutrophilic leukocytes and macrophages isolated from M-SCC, whereas the same fraction of peptides was always the highest, independent of leukocyte type, for cells recovered from H-SCC milk. In caprine milk, a level of 700,000 cells/mL represented the threshold for changes in leukocyte distribution, which is presumably related to the immune status of the mammary gland. Differences in the profile of

  16. Peptidomic analysis reveals proteolytic activity of kefir microorganisms on bovine milk proteins

    PubMed Central

    Dallas, David C.; Citerne, Florine; Tian, Tian; Silva, Vitor L. M.; Kalanetra, Karen M.; Frese, Steven A.; Robinson, Randall C.; Mills, David A.; Barile, Daniela

    2015-01-01

    Scope The microorganisms that make up kefir grains are well known for lactose fermentation, but the extent to which they hydrolyze and consume milk proteins remains poorly understood. Peptidomics technologies were used to examine the proteolytic activity of kefir grains on bovine milk proteins. Methods and results Gel electrophoresis revealed substantial digestion of milk proteins by kefir grains, with mass spectrometric analysis showing the release of 609 protein fragments and alteration of the abundance of >1,500 peptides that derived from 27 milk proteins. Kefir contained 25 peptides identified from the literature as having biological activity, including those with antihypertensive, antimicrobial, immunomodulatory, opioid and anti-oxidative functions. 16S rRNA and shotgun metagenomic sequencing identified the principle taxa in the culture as Lactobacillus species. Conclusion The model kefir sample contained thousands of protein fragments released in part by kefir microorganisms and in part by native milk proteases. PMID:26616950

  17. Peptidomic analysis reveals proteolytic activity of kefir microorganisms on bovine milk proteins.

    PubMed

    Dallas, David C; Citerne, Florine; Tian, Tian; Silva, Vitor L M; Kalanetra, Karen M; Frese, Steven A; Robinson, Randall C; Mills, David A; Barile, Daniela

    2016-04-15

    The microorganisms that make up kefir grains are well known for lactose fermentation, but the extent to which they hydrolyze and consume milk proteins remains poorly understood. Peptidomics technologies were used to examine the proteolytic activity of kefir grains on bovine milk proteins. Gel electrophoresis revealed substantial digestion of milk proteins by kefir grains, with mass spectrometric analysis showing the release of 609 protein fragments and alteration of the abundance of >1500 peptides that derived from 27 milk proteins. Kefir contained 25 peptides identified from the literature as having biological activity, including those with antihypertensive, antimicrobial, immunomodulatory, opioid and anti-oxidative functions. 16S rRNA and shotgun metagenomic sequencing identified the principle taxa in the culture as Lactobacillus species. The model kefir sample contained thousands of protein fragments released in part by kefir microorganisms and in part by native milk proteases. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. [Muriatic secretion and acid-proteolytic activity of the stomach in vivo].

    PubMed

    Gorshkov, V A; Zhigalova, T N; Avalueva, E B

    2005-01-01

    The study of the basal (BAP) and stimulated (SAP) acid production effect on the average daily acidity and speed of proteolysis in the stomach in vivo was conducted in 498 patients with various functional states of the stomach. It was established that the aforesaid muriatic secretion indices influence the acidity and speed of proteolysis in natural conditions of digestion only within the low and normal range of their values. In the condition of HCl hypersecretion, the dependence between the muriatic secretion and acidity and proteolytic stomach activity in vivo gets lost. This can be explained by the autoregulation effect supporting the introgastral pH optimal for pepsin activity irrespective of the intensity of the acid glands hyperplasia.

  19. Multiplexed homogeneous assays of proteolytic activity using a smartphone and quantum dots.

    PubMed

    Petryayeva, Eleonora; Algar, W Russ

    2014-03-18

    Semiconductor quantum dot (QD) bioconjugates, with their unique and highly advantageous physicochemical and optical properties, have been extensively utilized as probes for bioanalysis and continue to generate widespread interest for these applications. An important consideration for expanding the utility of QDs and making their use routine is to make assays with QDs more accessible for laboratories that do not specialize in nanomaterials. Here, we show that digital color imaging of QD photoluminescence (PL) with a smartphone camera is a viable, easily accessible readout platform for quantitative, multiplexed, and real-time bioanalyses. Red-, green-, and blue-emitting CdSeS/ZnS QDs were conjugated with peptides that were labeled with a deep-red fluorescent dye, Alexa Fluor 647, and the dark quenchers, QSY9 and QSY35, respectively, to generate Förster resonance energy transfer (FRET) pairs sensitive to proteolytic activity. Changes in QD PL caused by the activity of picomolar to nanomolar concentrations of protease were detected as changes in the red-green-blue (RGB) channel intensities in digital color images. Importantly, measurements of replicate samples made with smartphone imaging and a sophisticated fluorescence plate reader yielded the same quantitative results, including initial proteolytic rates and specificity constants. Homogeneous two-plex and three-plex assays for the activity of trypsin, chymotrypsin, and enterokinase were demonstrated with RGB imaging. Given the ubiquity of smartphones, this work largely removes any instrumental impediments to the adoption of QDs as routine tools for bioanalysis in research laboratories and is a critical step toward the use of QDs for point-of-care diagnostics. This work also adds to the growing utility of smartphones in analytical methods by enabling multiplexed fluorimetric assays within a single sample volume and across multiple samples in parallel.

  20. Targeting MALT1 Proteolytic Activity in Immunity, Inflammation and Disease: Good or Bad?

    PubMed

    Demeyer, Annelies; Staal, Jens; Beyaert, Rudi

    2016-02-01

    MALT1 is a signaling protein that plays a key role in immunity, inflammation, and lymphoid malignancies. For a long time MALT1 was believed to function as a scaffold protein, providing an assembly platform for other signaling proteins. This view changed dramatically when MALT1 was also found to have proteolytic activity and a capacity to fine-tune immune responses. Preclinical studies have fostered the belief that MALT1 is a promising therapeutic target in autoimmunity and B cell lymphomas. However, recent studies have shown that mice expressing catalytically-inactive MALT1 develop multi-organ inflammation and autoimmunity, and thus have tempered this initial enthusiasm. We discuss recent findings, highlighting the urgent need for a better mechanistic and functional understanding of MALT1 in host defense and disease.

  1. Activation of the heat-stable polypeptide of the ATP-dependent proteolytic system.

    PubMed Central

    Ciechanover, A; Heller, H; Katz-Etzion, R; Hershko, A

    1981-01-01

    It had been shown previously that the heat-stable polypeptide of the ATP-dependent proteolytic system of reticulocytes, designated APF-1, forms covalent conjugates with protein substrates in an ATP-requiring process. We now describe an enzyme that carries out the activation by ATP of the polypeptide with pyrophosphate displacement. The formation of AMP-polypeptide and transfer of the polypeptide to a secondary acceptor are suggested by an APF-1 requirement for ATP-PPi and ATP-AMP exchange reactions, respectively. With radiolabeled polypeptide, an ATP-dependent labeling of the enzyme was shown to be by a linkage that is acid stable but is labile to treatment with mild alkali, hydroxylamine, borohydride, or mercuric salts. It therefore appears that the AMP-polypeptide undergoes attack by an -SH group of the enzyme to form a thiolester. PMID:6262770

  2. Regulation of NUB1 Activity through Non-Proteolytic Mdm2-Mediated Ubiquitination

    PubMed Central

    Bonacci, Thomas; Audebert, Stéphane; Camoin, Luc; Baudelet, Emilie; Iovanna, Juan-Lucio

    2017-01-01

    NUB1 (Nedd8 ultimate buster 1) is an adaptor protein which negatively regulates the ubiquitin-like protein Nedd8 as well as neddylated proteins levels through proteasomal degradation. However, molecular mechanisms underlying this function are not completely understood. Here, we report that the oncogenic E3 ubiquitin ligase Mdm2 is a new NUB1 interacting protein which induces its ubiquitination. Interestingly, we found that Mdm2-mediated ubiquitination of NUB1 is not a proteolytic signal. Instead of promoting the conjugation of polyubiquitin chains and the subsequent proteasomal degradation of NUB1, Mdm2 rather induces its di-ubiquitination on lysine 159. Importantly, mutation of lysine 159 into arginine inhibits NUB1 activity by impairing its negative regulation of Nedd8 and of neddylated proteins. We conclude that Mdm2 acts as a positive regulator of NUB1 function, by modulating NUB1 ubiquitination on lysine 159. PMID:28099510

  3. A novel synthetic quinolinone inhibitor presents proteolytic and hemorrhagic inhibitory activities against snake venom metalloproteases.

    PubMed

    Baraldi, Patrícia T; Magro, Angelo J; Matioli, Fábio F; Marcussi, Silvana; Lemke, Ney; Calderon, Leonardo A; Stábeli, Rodrigo G; Soares, Andreimar M; Correa, Arlene G; Fontes, Marcos R M

    2016-02-01

    Metalloproteases play a fundamental role in snake venom envenomation inducing hemorrhagic, fibrigen(ogen)olytic and myotoxic effects in their victims. Several snake venoms, such as those from the Bothrops genus, present important local effects which are not efficiently neutralized by conventional serum therapy. Consequently, these accidents may result in permanent sequelae and disability, creating economic and social problems, especially in developing countries, leading the attention of the World Health Organization that considered ophidic envenomations a neglected tropical disease. Aiming to produce an efficient inhibitor against bothropic venoms, we synthesized different molecules classified as quinolinones - a group of low-toxic chemical compounds widely used as antibacterial and antimycobacterial drugs - and tested their inhibitory properties against hemorrhage caused by bothropic venoms. The results from this initial screening indicated the molecule 2-hydroxymethyl-6-methoxy-1,4-dihydro-4-quinolinone (Q8) was the most effective antihemorrhagic compound among all of the assayed synthetic quinolinones. Other in vitro and in vivo experiments showed this novel compound was able to inhibit significantly the hemorrhagic and/or proteolytic activities of bothropic crude venoms and isolated snake venom metalloproteases (SVMPs) even at lower concentrations. Docking and molecular dynamic simulations were also performed to get insights into the structural basis of Q8 inhibitory mechanism against proteolytic and hemorrhagic SVMPs. These structural studies demonstrated that Q8 may form a stable complex with SVMPs, impairing the access of substrates to the active sites of these toxins. Therefore, both experimental and structural data indicate that Q8 compound is an interesting candidate for antiophidic therapy, particularly for the treatment of the hemorrhagic and necrotic effects induced by bothropic venoms. Copyright © 2015 Elsevier B.V. and Société Française de

  4. Proteolytic activation of both components of the cation stress–responsive Slt pathway in Aspergillus nidulans

    PubMed Central

    Mellado, Laura; Arst, Herbert N.; Espeso, Eduardo A.

    2016-01-01

    Tolerance of Aspergillus nidulans to alkalinity and elevated cation concentrations requires both SltA and SltB. Transcription factor SltA and the putative pseudokinase/protease signaling protein SltB comprise a regulatory pathway specific to filamentous fungi. In vivo, SltB is proteolytically cleaved into its two principal domains. Mutational analysis defines a chymotrypsin-like serine protease domain that mediates SltB autoproteolysis and proteolytic cleavage of SltA. The pseudokinase domain might modulate the protease activity of SltB. Three forms of the SltA transcription factor coexist in cells: a full-length, 78-kDa version and a processed, 32-kDa form, which is found in phosphorylated and unphosphorylated states. The SltA32kDa version mediates transcriptional regulation of sltB and, putatively, genes required for tolerance to cation stress and alkalinity. The full-length form, SltA78kDa, apparently has no transcriptional function. In the absence of SltB, only the primary product of SltA is detectable, and its level equals that of SltA78kDa. Mutations in sltB selected as suppressors of null vps alleles and resulting in cation/alkalinity sensitivity either reduced or eliminated SltA proteolysis. There is no evidence for cation or alkalinity regulation of SltB cleavage, but activation of sltB expression requires SltA. This work identifies the molecular mechanisms governing the Slt pathway. PMID:27307585

  5. Proteolytic activation of both components of the cation stress-responsive Slt pathway in Aspergillus nidulans.

    PubMed

    Mellado, Laura; Arst, Herbert N; Espeso, Eduardo A

    2016-08-15

    Tolerance of Aspergillus nidulans to alkalinity and elevated cation concentrations requires both SltA and SltB. Transcription factor SltA and the putative pseudokinase/protease signaling protein SltB comprise a regulatory pathway specific to filamentous fungi. In vivo, SltB is proteolytically cleaved into its two principal domains. Mutational analysis defines a chymotrypsin-like serine protease domain that mediates SltB autoproteolysis and proteolytic cleavage of SltA. The pseudokinase domain might modulate the protease activity of SltB. Three forms of the SltA transcription factor coexist in cells: a full-length, 78-kDa version and a processed, 32-kDa form, which is found in phosphorylated and unphosphorylated states. The SltA32kDa version mediates transcriptional regulation of sltB and, putatively, genes required for tolerance to cation stress and alkalinity. The full-length form, SltA78kDa, apparently has no transcriptional function. In the absence of SltB, only the primary product of SltA is detectable, and its level equals that of SltA78kDa. Mutations in sltB selected as suppressors of null vps alleles and resulting in cation/alkalinity sensitivity either reduced or eliminated SltA proteolysis. There is no evidence for cation or alkalinity regulation of SltB cleavage, but activation of sltB expression requires SltA. This work identifies the molecular mechanisms governing the Slt pathway. © 2016 Mellado et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  6. Proteolytic profiling and comparative analyses of active trypsin-like serine peptidases in preimaginal stages of Culex quinquefasciatus

    PubMed Central

    2012-01-01

    Background The mosquito Culex quinquefasciatu s, a widespread insect in tropical and sub-tropical regions of the world, is a vector of multiple arboviruses and parasites, and is considered an important risk to human and veterinary health. Proteolytic enzymes play crucial roles in the insect physiology including the modulation of embryonic development and food digestion. Therefore, these enzymes represent important targets for the development of new control strategies. This study presents zymographic characterization and comparative analysis of the proteolytic activity found in eggs, larval instars and pupae of Culex quinquefasciatus. Methods The proteolytic profiles of eggs, larvae and pupa of Cx. quinquefasciatus were characterized by SDS-PAGE co-polymerized with 0.1% gelatin, according to the pH, temperature and peptidase inhibitor sensitivity. In addition, the proteolytic activities were characterized in solution using 100 μM of the fluorogenic substrate Z-Phe-Arg-AMC. Results Comparison of the proteolytic profiles by substrate-SDS-PAGE from all preimaginal stages of the insect revealed qualitative and quantitative differences in the peptidase expression among eggs, larvae and pupae. Use of specific inhibitors revealed that the proteolytic activity from preimaginal stages is mostly due to trypsin-like serine peptidases that display optimal activity at alkaline pH. In-solution, proteolytic assays of the four larval instars using the fluorogenic substrate Z-Phe-Arg-AMC in the presence or absence of a trypsin-like serine peptidase inhibitor confirmed the results obtained by substrate-SDS-PAGE analysis. The trypsin-like serine peptidases of the four larval instars were functional over a wide range of temperatures, showing activities at 25°C and 65°C, with an optimal activity between 37°C and 50°C. Conclusion The combined use of zymography and in-solution assays, as performed in this study, allowed for a more detailed analysis of the repertoire of proteolytic

  7. The proteolytic system of pineapple stems revisited: Purification and characterization of multiple catalytically active forms.

    PubMed

    Matagne, André; Bolle, Laetitia; El Mahyaoui, Rachida; Baeyens-Volant, Danielle; Azarkan, Mohamed

    2017-02-23

    Crude pineapple proteases extract (aka stem bromelain; EC 3.4.22.4) is an important proteolytic mixture that contains enzymes belonging to the cysteine proteases of the papain family. Numerous studies have been reported aiming at the fractionation and characterization of the many molecular species present in the extract, but more efforts are still required to obtain sufficient quantities of the various purified protease forms for detailed physicochemical, enzymatic and structural characterization. In this work, we describe an efficient strategy towards the purification of at least eight enzymatic forms. Thus, following rapid fractionation on a SP-Sepharose FF column, two sub-populations with proteolytic activity were obtained: the unbound (termed acidic) and bound (termed basic) bromelain fractions. Following reversible modification with monomethoxypolyethylene glycol (mPEG), both fractions were further separated on Q-Sepharose FF and SP-Sepharose FF, respectively. This procedure yielded highly purified molecular species, all titrating ca. 1 mol of thiol group per mole of enzyme, with distinct biochemical properties. N-terminal sequencing allowed identifying at least eight forms with proteolytic activity. The basic fraction contained previously identified species, i.e. basic bromelain forms 1 and 2, ananain forms 1 and 2, and comosain (MEROPS identifier: C01.027). Furthermore, a new proteolytic species, showing similarities with basic bomelain forms 1 and 2, was discovered and termed bromelain form 3. The two remaining species were found in the acidic bromelain fraction and were arbitrarily named acidic bromelain forms 1 and 2. Both, acidic bromelain forms 1, 2 and basic bromelain forms 1, 2 and 3 are glycosylated, while ananain forms 1 and 2, and comosain are not. The eight protease forms display different amidase activities against the various substrates tested, namely small synthetic chromogenic compounds (DL-BAPNA and Boc-Ala-Ala-Gly-pNA), fluorogenic compounds

  8. Low-volume multiplexed proteolytic activity assay and inhibitor analysis through a pico-injector array.

    PubMed

    Ng, Ee Xien; Miller, Miles A; Jing, Tengyang; Lauffenburger, Doug A; Chen, Chia-Hung

    2015-02-21

    Secreted active proteases, from families of enzymes such as matrix metalloproteinases (MMPs) and ADAMs (a disintegrin and metalloproteinases), participate in diverse pathological processes. To simultaneously measure multiple specific protease activities, a series of parallel enzyme reactions combined with a series of inhibitor analyses for proteolytic activity matrix analysis (PrAMA) are essential but limited due to the sample quantity requirements and the complexity of performing multiple reactions. To address these issues, we developed a pico-injector array to generate 72 different reactions in picoliter-volume droplets by controlling the sequence of combinational injections, which allowed simultaneous recording of a wide range of multiple enzyme reactions and measurement of inhibitor effects using small sample volumes (~10 μL). Multiple MMP activities were simultaneously determined by 9 different substrates and 2 inhibitors using injections from a pico-injector array. Due to the advantages of inhibitor analysis, the MMP/ADAM activities of MDA-MB-231, a breast cancer cell line, were characterized with high MMP-2, MMP-3 and ADAM-10 activity. This platform could be customized for a wide range of applications that also require multiple reactions with inhibitor analysis to enhance the sensitivity by encapsulating different chemical sensors.

  9. Bioactive peptides from caseins released by cold active proteolytic enzymes from Arsukibacterium ikkense.

    PubMed

    De Gobba, Cristian; Tompa, Gorazd; Otte, Jeanette

    2014-12-15

    Proteolytic enzymes secreted by the cold-adapted microorganism Arsukibacterium ikkense were tested for their ability to degrade caseins at low temperature and produce bioactive peptides. The caseins were extensively degraded (90%) after 24h of hydrolysis at 5°C and completely degraded at 25°C, and many novel peptides were formed. The most hydrolysed sample showed high angiotensin I converting enzyme (ACE)-inhibitory and antioxidant activity, and a number of potent ACE-inhibitory and antioxidant peptides were identified. The presence of tyrosine seemed fundamental for both ACE-inhibitory and antioxidant activity, while phenylalanine seemed to potentiate the antioxidant activity. The novel peptide YPELF was found to have strong radical scavenging and lipid oxidation inhibitory activities, with IC50 for both around 3.5μM. None of the hydrolysates showed antimicrobial activity. Secreted enzymes from cultures of A. ikkense could thus be a valuable enzyme preparation for inexpensive, energy-efficient production of potent bioactive peptides from caseins in milk at low temperatures. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. The microenvironment patterns the pluripotent mouse epiblast through paracrine Furin and Pace4 proteolytic activities.

    PubMed

    Mesnard, Daniel; Donnison, Martyn; Fuerer, Christophe; Pfeffer, Peter L; Constam, Daniel B

    2011-09-01

    The fate of pluripotent cells in early mouse embryos is controlled by graded Nodal signals that are activated by the endoproteases Furin and Pace4. Soluble forms of Furin and Pace4 cleave proNodal in vitro and after secretion in transfected cells, but direct evidence for paracrine activity in vivo is elusive. Here, we show that Furin and Pace4 are released by the extraembryonic microenvironment, and that they cleave a membrane-bound reporter substrate in adjacent epiblast cells and activate Nodal to maintain pluripotency. Secreted Pace4 and Furin also stimulated mesoderm formation, whereas endoderm was only induced by Pace4, correlating with a difference in the spatiotemporal distribution of these proteolytic activities. Our analysis of paracrine Furin and Pace4 activities and their in vivo functions significantly advances our understanding of how the epiblast is patterned by its microenvironment. Adding cell-cell communication to the pleiotropic portfolio of these proteases provides a new framework to study proprotein processing also in other relevant contexts.

  11. Interactions between Lactobacillus sakei and CNC (Staphylococcus xylosus and Kocuria varians) and their influence on proteolytic activity.

    PubMed

    Tremonte, P; Reale, A; Di Renzo, T; Tipaldi, L; Di Luccia, A; Coppola, R; Sorrentino, E; Succi, M

    2010-11-01

    To evaluate interactions between Lactobacillus sakei and coagulase negative cocci (CNC) (Staphylococcus xylosus and Kocuria varians) and to investigate the influence of these interactions on their own proteolytic activity. Interactions occurring between strains of Lact. sakei and CNC were assessed by spectrophotometric analysis. The growth of 35 strains of Lact. sakei, used as indicators, was compared to that obtained combining the same strains with growing cells or cell-free supernatants of 20 CNC (18 Staph. xylosus and 2 K. varians). The proteolytic activity expressed by single strains or by their combinations was assessed on sarcoplasmic protein extracts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results evidenced that interactions are able to affect not only the growth but also the in vitro proteolytic activity of Lact. sakei and CNC used in combination. A relationship between the presence of interactions among useful strains and the strength of technological characteristics, such as proteolysis, was defined. The study highlighted that CNC are able to stimulate the growth of some Lact. sakei strains. At the same time, this interaction positively influences the proteolytic activity of strains used in combination. Given the importance of proteolysis during the ripening of fermented meats, this phenomenon should be taken into account to select meat starter cultures. © 2010 The Authors. © 2010 The Society for Applied Microbiology.

  12. Effects of chymostatin and other proteinase inhibitors on protein breakdown and proteolytic activities in muscle

    PubMed Central

    Libby, Peter; Goldberg, Alfred L.

    1980-01-01

    To learn more about the enzymes involved in protein catabolism in skeletal and cardiac muscle and to identify selective inhibitors of this process, we studied the effects of proteinase inhibitors on protein turnover in isolated muscles and on proteolytic activities in muscle homogenates. Chymostatin (20μm) decreased protein breakdown by 20–40% in leg muscles from normal rodents and also in denervated and dystrophic muscles. These results are similar to our previous findings with leupeptin. The related inhibitors pepstatin, bestatin, and elastatinal did not decrease protein breakdown; antipain slowed this process in rat hind-limb muscles but not in diaphragm. Chymostatin did not decrease protein synthesis and thus probably retards proteolysis by a specific effect on cell proteinase(s). In homogenates of rat muscle, chymostatin, in common with leupeptin and antipain, inhibits the lysosomal proteinase cathepsin B, and the soluble Ca2+-activated proteinase. In addition, chymostatin, but not leupeptin, inhibits the chymotrypsin-like proteinase apparent in muscle homogenates. In muscles depleted of most of this activity by treatment with the mast-cell-degranulating agent 48/80, chymostatin still decreased protein breakdown. Therefore inhibition of this alkaline activity probably does not account for the decrease in protein breakdown. These results are consistent with a lysosomal site of action for chymostatin. Because of its lack of toxicity, chymostatin may be useful in maintaining tissues in vitro and perhaps in decreasing muscle atrophy in vivo. PMID:7406880

  13. Bioactive peptides encrypted in milk proteins: proteolytic activation and thropho-functional properties.

    PubMed

    Meisel, H; Bockelmann, W

    1999-01-01

    The bioactivities of peptides encrypted in major milk proteins are latent until released and activated by enzymatic proteolysis, e.g. during gastrointestinal digestion or food processing. The proteolytic system of lactic acid bacteria can contribute to the liberation of bioactive peptides. In vitro, the purified cell wall proteinase of Lactococcus lactis was shown to liberate oligopeptides from beta- and alpha-caseins which contain amino acid sequences present in casomorphins, casokinines, and immunopeptides. The further degradation of these peptides by endopeptidases and exopeptidases of lactic acid bacteria could lead to the liberation of bioactive peptides in fermented milk products. However, the sequences of practically all known biologically active peptides can also be cleaved by peptidases from lactic acid bacteria. Activated peptides are potential modulators of various regulatory processes in the body: Opioid peptides are opioid receptor ligands which can modulate absorption processes in the intestinal tract, angiotensin-I-converting enzyme (ACE)-inhibitory peptides are hemodynamic regulators and exert an antihypertensive effect, immunomodulating casein peptides stimulate the activities of cells of the immune system, antimicrobial peptides kill sensitive microorganisms, antithrombotic peptides inhibit aggregation of platelets and caseinophosphopeptides may function as carriers for different minerals, especially calcium. Bioactive peptides can interact with target sites at the luminal side of the intestinal tract. Furthermore, they can be absorbed and then reach peripheral organs. Food-derived bioactive peptides are claimed to be health enhancing components which can be used for functional food and pharmaceutical preparations.

  14. Differential proteolytic activation of factor VIII-von Willebrand factor complex by thrombin

    SciTech Connect

    Hill-Eubanks, D.C.; Parker, C.G.; Lollar, P. )

    1989-09-01

    Blood coagulation factor VIII (fVIII) is a plasma protein that is decreased or absent in hemophilia A. It is isolated as a mixture of heterodimers that contain a variably sized heavy chain and a common light chain. Thrombin catalyzes the activation of fVIII in a reaction that is associated with cleavages in both types of chain. The authors isolated a serine protease from Bothrops jararacussu snake venom that catalyzes thrombin-like heavy-chain cleavage but not light-chain cleavage in porcine fVIII as judged by NaDodSO{sub 4}/PAGE and N-terminal sequence analysis. Using a plasma-free assay of the ability of activated {sup 125}I-fVIII to function as a cofactor in the activation of factor X by factor IXa, they found that fVIII is activated by the venom enzyme. The venom enzyme-activated fVIII was isolated in stable form by cation-exchange HPLC. von Willebrand factor inhibited venom enzyme-activated fVIII but not thrombin-activated fVIII. These results suggest that the binding of fVIII to von Willebrand factor depends on the presence of an intact light chain and that activated fVIII must dissociate from von Willebrand factor to exert its cofactor effect. Thus, proteolytic activation of fVIII-von Willebrand factor complex appears to be differentially regulated by light-chain cleavage to dissociate the complex and heavy-chain cleavage to activate the cofactor function.

  15. Protease Gene Duplication and Proteolytic Activity in Drosophila Female Reproductive Tracts

    PubMed Central

    Kelleher, Erin S.; Pennington, James E.

    2009-01-01

    Secreted proteases play integral roles in sexual reproduction in a broad range of taxa. In the genetic model Drosophila melanogaster, these molecules are thought to process peptides and activate enzymes inside female reproductive tracts, mediating critical postmating responses. A recent study of female reproductive tract proteins in the cactophilic fruit fly Drosophila arizonae, identified pervasive, lineage-specific gene duplication amongst secreted proteases. Here, we compare the evolutionary dynamics, biochemical nature, and physiological significance of secreted female reproductive serine endoproteases between D. arizonae and its congener D. melanogaster. We show that D. arizonae lower female reproductive tract (LFRT) proteins are significantly enriched for recently duplicated secreted proteases, particularly serine endoproteases, relative to D. melanogaster. Isolated lumen from D. arizonae LFRTs, furthermore, exhibits significant trypsin-like and elastase-like serine endoprotease acitivity, whereas no such activity is seen in D. melanogaster. Finally, trypsin- and elastase-like activity in D. arizonae female reproductive tracts is negatively regulated by mating. We propose that the intense proteolytic environment of the D. arizonae female reproductive tract relates to the extraordinary reproductive physiology of this species and that ongoing gene duplication amongst these proteases is an evolutionary consequence of sexual conflict. PMID:19546158

  16. Effect of wine inhibitors on the proteolytic activity of papain from Carica papaya L. latex.

    PubMed

    Benucci, Ilaria; Esti, Marco; Liburdi, Katia

    2015-01-01

    The influence of potential inhibitors naturally present in wine on the proteolytic activity of papain from Carica papaya latex was investigated to evaluate its applicability in white wine protein haze stabilization. Enzymatic activity was tested against a synthetic tripeptide chromogenic substrate in wine-like acidic medium that consisted of tartaric buffer (pH 3.2) supplemented with ethanol, free sulfur dioxide (SO2 ), grape skin and seed tannins within the average ranges of concentrations that are typical in wine. The diagnosis of inhibition type, performed with the graphical method, demonstrated that all of tested wine constituents were reversible inhibitors of papain. The strongest inhibition was exerted by free SO2 , which acted as a mixed-type inhibitor, similar to grape skin and seed tannins. Finally, when tested in table white wines, the catalytic activity of papain, even when if it was ascribable to the hyperbolic behavior of Michaelis-Menten equation, was determined to be strongly affected by free SO2 and total phenol level. © 2014 American Institute of Chemical Engineers.

  17. Proteolytic degradation of intestinal mucosal extracellular matrix after lamina propria T cell activation.

    PubMed Central

    Pender, S L; Lionetti, P; Murch, S H; Wathan, N; MacDonald, T T

    1996-01-01

    BACKGROUND: Proteoglycans, consisting of glycosaminoglycan (GAG) side chains covalently linked to a protein core, are a major component of the extracellular matrix of the intestinal lamina propria. AIMS: This study investigated the effects of lamina propria T cell activation on the proteoglycan component of the matrix. METHODS: The high degree of sulphation of GAGs means that they are polyanionic and thus can be visualised in tissue sections by means of colloidal-gold labelled cationic probes. RESULTS: In human fetal small intestine there is a dense meshwork of anionic residues in the lamina propria and basement membrane. When explants of human fetal small intestine are cultured ex vivo, and resident lamina propria T cells are activated with pokeweed mitogen, mucosal destruction occurs within three days. This is associated with the rapid loss of anionic sites from the lamina propria. Dermatan sulphate proteoglycan is lost from the tissue and is present at increased concentrations in the organ culture supernatants, indicating that T cell activation has led to solubilisation of lamina propria proteoglycans. Tissue destruction and loss of anionic residues are inhibited in a dose dependent fashion by dexamethasone, and by the protease inhibitor, alpha 2 macroglobulin. CONCLUSIONS: Proteolytic degradation of the lamina propria may therefore be a mechanism by which T cell hypersensitivity injures the intestinal mucosa. Images Figure 1 Figure 4 Figure 5 PMID:8977345

  18. Proteolytic Activity at Alkaline pH in Oat Leaves, Isolation of an Aminopeptidase 1

    PubMed Central

    Casano, Leonardo M.; Desimone, Marcelo; Trippi, Victorio S.

    1989-01-01

    Proteolytic activity in oat leaf extracts was measured with both azocasein and ribulose bisphosphate carboxylase (Rubisco) as substrates over a wide range of pH (3.0-9.2). With either azocasein or Rubisco activity peaks appeared at pH 4.8, 6.6, and 8.4. An aminopeptidase (AP) which hydrolyzes leucine-nitroanilide was partially purified. Purification consisted of a series of six steps which included ammonium sulfate precipitation, gel filtration, and two ionic exchange chromatographies. The enzyme was purified more than 100-fold. The apparent Km for leucine-nitroanilide is 0.08 millimolar at its pH optimum of 8.4. AP may be a cystein protease since it is inhibited by heavy metals and activated by 2-mercaptoethanol. Isolated chloroplasts were also able to hydrolyze leucine-nitroanilide at a pH optimum of 8.4, indicating that AP could be localized inside the photosynthetic organelles. PMID:16667194

  19. Studies on the membrane-associated proteolytic activities of Escherichia coli

    SciTech Connect

    Palmer, S.M.

    1986-01-01

    The membrane fraction contains three proteolytic activities which can be resolved from whole membrane detergent extracts by DEAE-cellulose chromatography. The first two eluting activities have been previously reported as protease V and protease IV. These two enzymes were further purified by gel permeation HPLC. Protease V has a M. W. of 31,000 in SDS-PAGE gels. Protease IV has a M. W. of 62,000 and exists in two distinct isoforms of pl approx. = 6.7 and 6.9. The third enzyme eluting from the DEAE-cellulose column was further purified by affinity chromatography on Benzamidine-Sepharose 6B. This enzyme, referred to herein as protease VI, is a 43 kdal protein which has not been previously characterized. Protease VI was sensitive to inhibition by the serine protease inhibitors phenylmethylsulfony fluoride (PMSF), diisopropylfluorophosphate (DFP) and p-amino-benzamidine (PAB). Additionally, this enzyme exhibited maximal activity at pH approx. = 8.0. Incubation of whole membrane preparations with (/sup 3/H) DFP resulted in 11 specific proteins acquiring the radioactive label, included in this group of proteins were proteases IV, V, and VI. Several of the DFP-reactive proteins were also shown to bind (/sup 125/I) ampicillin.

  20. Conservation of intramembrane proteolytic activity and substrate specificity in prokaryotic and eukaryotic rhomboids.

    PubMed

    Urban, Sinisa; Schlieper, Daniel; Freeman, Matthew

    2002-09-03

    Rhomboid is an intramembrane serine protease responsible for the proteolytic activation of Drosophila epidermal growth factor receptor (EGFR) ligands. Although nothing is known about the function of the approximately 100 currently known rhomboid genes conserved throughout evolution, a recent analysis suggests that a Rhomboid from the pathogenic bacterium Providencia stuartii is involved in the production of a quorum-sensing factor. This suggests that an intercellular signaling mechanism may have been conserved between prokaryotes and metazoans. However, the function of prokaryotic Rhomboids is unknown. We have examined the ability of eight prokaryotic Rhomboids to cleave the three Drosophila EGFR ligands. Despite their striking sequence divergence, Rhomboids from one Gram-positive and four Gram-negative species, including Providencia, specifically cleaved Drosophila substrates, but not similar proteins such as Transforming Growth Factor alpha (TGFalpha) and Delta. Although the sequence similarity between these divergent Rhomboids is very limited, all contain the putative serine catalytic triad residues, and their specific mutation abolished protease activity. Therefore, despite low overall homology, the Rhomboids are a family of ancient, functionally conserved intramembrane serine proteases, some of which also have conserved substrate specificity. Moreover, a function for Rhomboids in activating intercellular signaling appears to have evolved early.

  1. Bacterial profiles and proteolytic activity in peri-implantitis versus healthy sites.

    PubMed

    Neilands, J; Wickström, C; Kinnby, B; Davies, J R; Hall, J; Friberg, B; Svensäter, G

    2015-10-01

    Peri-implantitis is a biofilm-induced destructive inflammatory process that, over time, results in loss of supporting bone around an osseointegrated dental implant. Biofilms at peri-implantitis sites have been reported to be dominated by Gram-negative anaerobic rods with a proteolytic metabolism such as, Fusobacterium, Porphyromonas, Prevotella and Tannerella, as well as anaerobic Gram-positive cocci. In this study, we hypothesized that protease activity is instrumental in driving bone destruction and we therefore compared the microbial composition and level of protease activity in samples of peri-implant biofluid (PIBF) from 25 healthy subjects (H group) and 25 subjects with peri-implantitis (PI group). Microbial composition was investigated using culture techniques and protease activity was determined using a FITC-labelled casein substrate. The microbial composition was highly variable in subjects both in the H and PI groups but one prominent difference was the prevalence of Porphyromonas/Prevotella and anaerobic Gram positive cocci which was significantly higher in the PI than in the H group. A subgroup of subjects with peri-implantitis displayed a high level of protease activity in the PIBF compared to healthy subjects. However, this activity could not be related to the presence of specific bacterial species. We propose that a high level of protease activity may be a predictive factor for disease progression in peri-implantitis. Further longitudinal studies are however required to determine whether assessment of protease activity could serve as a useful method to identify patients at risk for progressive tissue destruction. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Cysteine protease gene expression and proteolytic activity during senescence of Alstroemeria petals.

    PubMed

    Wagstaff, Carol; Leverentz, Michael K; Griffiths, Gareth; Thomas, Brian; Chanasut, Usawadee; Stead, Anthony D; Rogers, Hilary J

    2002-02-01

    The functional life of the flower is terminated by senescence and/or abscission. Multiple processes contribute to produce the visible signs of petal wilting and inrolling that typify senescence, but one of the most important is that of protein degradation and remobilization. This is mediated in many species through protein ubiquitination and the action of specific protease enzymes. This paper reports the changes in protein and protease activity during development and senescence of Alstroemeria flowers, a Liliaceous species that shows very little sensitivity to ethylene during senescence and which shows perianth abscission 8-10 d after flower opening. Partial cDNAs of ubiquitin (ALSUQ1) and a putative cysteine protease (ALSCYP1) were cloned from Alstroemeria using degenerate PCR primers and the expression pattern of these genes was determined semi-quantitatively by RT-PCR. While the levels of ALSUQ1 only fluctuated slightly during floral development and senescence, there was a dramatic increase in the expression of ALSCYP1 indicating that this gene may encode an important enzyme for the proteolytic process in this species. Three papain class cysteine protease enzymes showing different patterns of activity during flower development were identified on zymograms, one of which showed a similar expression pattern to the cysteine protease cDNA.

  3. Functional characterization of the proteolytic activity of the tomato black ring nepovirus RNA-1-encoded polyprotein.

    PubMed

    Hemmer, O; Greif, C; Dufourcq, P; Reinbolt, J; Fritsch, C

    1995-01-10

    Translation of tomato black ring virus (TBRV) RNA-1 in a rabbit reticulocyte lysate leads to the synthesis of a 250K polyprotein which cleaves itself into smaller proteins of 50, 60, 120, and 190K. Polypeptides synthesized from synthetic transcripts corresponding to different regions of TBRV RNA-1 are processed only when they encode the 23K protein delimited earlier by sequence homology with the cowpea mosaic virus 24K protease. The proteolytic activity of this protein is completely lost by mutating residues C170 (to I) or L188 (to H), residues which align with conserved residues of the viral serine-like proteases. The 120K protein is generated by cleavage of the dipeptide K/A localized in front of the VPg but is not further cleaved in vitro at the K/S site (at the C terminus of the VPg) or between the protease and polymerase domains. However, both the protein VPgProPol (120K) and the protein ProPol (117K) produced in vitro from synthetic transcripts can cleave in trans the RNA-2-encoded 150K polyprotein, but they cannot cleave in trans polypeptides containing a cleavage site expressed from RNA-1 transcripts in which the protease cistron is absent or modified.

  4. A plant Bcl-2-associated athanogene is proteolytically activated to confer fungal resistance

    PubMed Central

    Kabbage, Mehdi; Kessens, Ryan; Dickman, Martin B.

    2016-01-01

    The Bcl-2-associated athanogene (BAG) family is a multifunctional group of proteins involved in numerous cellular functions ranging from apoptosis to tumorigenesis. These proteins are evolutionarily conserved and encode a characteristic region known as the BAG domain. BAGs function as adapter proteins forming complexes with signaling molecules and molecular chaperones. In humans, a role for BAG proteins has been suggested in tumor growth, HIV infection, and neurodegenerative diseases; as a result, the BAGs are attractive targets for therapeutic interventions, and their expression in cells may serve as a predictive tool for disease development. The Arabidopsis genome contains seven homologs of BAG family proteins (Figure 1), including four with a domain organization similar to animal BAGs (BAG1-4). The remaining three members (BAG5-7) contain a predicted calmodulin-binding motif near the BAG domain, a feature unique to plant BAG proteins that possibly reflects divergent mechanisms associated with plant-specific functions. As reported for animal BAGs, plant BAGs also regulate several stress and developmental processes (Figure 2). The recent article by Li et al. focuses on the role of BAG6 in plant innate immunity. This study shows that BAG6 plays a key role in basal plant defense against fungal pathogens. Importantly, this work further shows that BAG6 is proteolytically activated to induce autophagic cell death and resistance in plants. This finding underscores the importance of proteases in the execution of plant cell death, yet little is known about proteases and their substrates in plants. PMID:28358147

  5. Proteasome inhibitor-induced apoptosis is mediated by positive feedback amplification of PKCδ proteolytic activation and mitochondrial translocation

    PubMed Central

    Sun, Faneng; Kanthasamy, Arthi; Song, Chunjuan; Yang, Yongjie; Anantharam, Vellareddy; Kanthasamy, Anumantha G

    2008-01-01

    Emerging evidence implicates impaired protein degradation by the ubiquitin proteasome system (UPS) in Parkinson's disease; however cellular mechanisms underlying dopaminergic degeneration during proteasomal dysfunction are yet to be characterized. In the present study, we identified that the novel PKC isoform PKCδ plays a central role in mediating apoptotic cell death following UPS dysfunction in dopaminergic neuronal cells. Inhibition of proteasome function by MG-132 in dopaminergic neuronal cell model (N27 cells) rapidly depolarized mitochondria independent of ROS generation to activate the apoptotic cascade involving cytochrome c release, and caspase-9 and caspase-3 activation. PKCδ was a key downstream effector of caspase-3 because the kinase was proteolytically cleaved by caspase-3 following exposure to proteasome inhibitors MG-132 or lactacystin, resulting in a persistent increase in the kinase activity. Notably MG-132 treatment resulted in translocation of proteolytically cleaved PKCδ fragments to mitochondria in a time-dependent fashion, and the PKCδ inhibition effectively blocked the activation of caspase-9 and caspase-3, indicating that the accumulation of the PKCδ catalytic fragment in the mitochondrial fraction possibly amplifies mitochondria-mediated apoptosis. Overexpression of the kinase active catalytic fragment of PKCδ (PKCδ-CF) but not the regulatory fragment (RF), or mitochondria-targeted expression of PKCδ-CF triggers caspase-3 activation and apoptosis. Furthermore, inhibition of PKCδ proteolytic cleavage by a caspase-3 cleavage-resistant mutant (PKCδ-CRM) or suppression of PKCδ expression by siRNA significantly attenuated MG-132-induced caspase-9 and -3 activation and DNA fragmentation. Collectively, these results demonstrate that proteolytically activated PKCδ has a significant feedback regulatory role in amplification of the mitochondria-mediated apoptotic cascade during proteasome dysfunction in dopaminergic neuronal cells. PMID

  6. Seasonal changes in proteolytic activity of calpains in striated muscles of long-tailed ground squirrel Spermophilus undulatus.

    PubMed

    Popova, S S; Vikhlyantsev, I M; Zakharova, N M; Podlubnaya, Z A; Fesenko, E E

    2017-01-01

    Seasonal changes in proteolytic activity and content of calpains in striated muscles of the longtailed ground squirrel Spermophilus undulatus were studied by casein zymography and Western blotting analysis. The results testify to hyperactivation of calpain proteases in the skeletal muscles of awakened animals during the "winter" activity. The observed changes are discussed in the context of adaptation of skeletal muscles of long-tailed ground squirrels to hibernation.

  7. Plant proteolytic enzyme papain abrogates angiogenic activation of human umbilical vein endothelial cells (HUVEC) in vitro

    PubMed Central

    2013-01-01

    Background Vascular endothelial growth factor (VEGF) is a key regulator of physiologic and pathogenic angiogenesis in diseases such as cancer and diabetic retinopathy. It is known that cysteine proteases from plants, like bromelain and papain are capable to suppress inflammatory activation. Recent studies have demonstrated that they may interfere with angiogenesis related pathways as well. The aim of this study was to investigate the anti-angiogenic effects of papain on human umbilical vein endothelial cells (HUVEC) in vitro. Methods Cell viability after prolonged treatment with papain was investigated by life cell staining and lactate dehydrogenase release assay. Angiogenic activation was assessed by ELISA against phosphorylated proteins AKT, MEK1/2, ERK1/2, SAPK/JNK and p38-MAPK. Growth inhibition was determined by means of an MTT-assay and cell migration by means of a scratch assay. Capability to form a capillary network was investigated using a tube formation assay. Results Papain did not induce proteolysis or cell detachment of HUVEC in a concentration range between 0 and 25 μg/mL. Four hours treatment with 10 μg/mL papain resulted in a reduced susceptibility of endothelial cells to activation by VEGF as determined by phosphorylation levels of Akt, MEK1/2, SAPK/JNK. Papain exerted a distinct inhibitory effect on cell growth, cell migration and tube formation with inhibition of tube formation detectable at concentrations as low as 1 μg/mL. Bromelain and ficin displayed similar effects with regard to cell growth and tube formation. Conclusion Papain showed a strong anti-angiogenic effect in VEGF activated HUVEC. This effect may be due to interference with AKT, MEK1/2 and SAPK/JNK phosphorylation. Two other plant derived cysteine proteases displayed similar inhibition of HUVEC cell growth and tube formation. These findings indicate that plant proteolytic enzymes may have potential as preventive and therapeutic agents against angiogenesis related human diseases

  8. Matriptase proteolytically activates influenza virus and promotes multicycle replication in the human airway epithelium.

    PubMed

    Beaulieu, Alexandre; Gravel, Émilie; Cloutier, Alexandre; Marois, Isabelle; Colombo, Éloïc; Désilets, Antoine; Verreault, Catherine; Leduc, Richard; Marsault, Éric; Richter, Martin V

    2013-04-01

    Influenza viruses do not encode any proteases and must rely on host proteases for the proteolytic activation of their surface hemagglutinin proteins in order to fuse with the infected host cells. Recent progress in the understanding of human proteases responsible for influenza virus hemagglutinin activation has led to the identification of members of the type II transmembrane serine proteases TMPRSS2 and TMPRSS4 and human airway trypsin-like protease; however, none has proved to be the sole enzyme responsible for hemagglutinin cleavage. In this study, we identify and characterize matriptase as an influenza virus-activating protease capable of supporting multicycle viral replication in the human respiratory epithelium. Using confocal microscopy, we found matriptase to colocalize with hemagglutinin at the apical surface of human epithelial cells and within endosomes, and we showed that the soluble form of the protease was able to specifically cleave hemagglutinins from H1 virus, but not from H2 and H3 viruses, in a broad pH range. We showed that small interfering RNA (siRNA) knockdown of matriptase in human bronchial epithelial cells significantly blocked influenza virus replication in these cells. Lastly, we provide a selective, slow, tight-binding inhibitor of matriptase that significantly reduces viral replication (by 1.5 log) of H1N1 influenza virus, including the 2009 pandemic virus. Our study establishes a three-pronged model for the action of matriptase: activation of incoming viruses in the extracellular space in its shed form, upon viral attachment or exit in its membrane-bound and/or shed forms at the apical surface of epithelial cells, and within endosomes by its membrane-bound form where viral fusion takes place.

  9. Proteolytic Processing and Activation of Clostridium perfringens Epsilon Toxin by Caprine Small Intestinal Contents

    PubMed Central

    Freedman, John C.; Li, Jihong; Uzal, Francisco A.

    2014-01-01

    ABSTRACT Epsilon toxin (ETX), a pore-forming toxin produced by type B and D strains of Clostridium perfringens, mediates severe enterotoxemia in livestock and possibly plays a role in human disease. During enterotoxemia, the nearly inactive ETX prototoxin is produced in the intestines but then must be activated by proteolytic processing. The current study sought to examine ETX prototoxin processing and activation ex vivo using the intestinal contents of a goat, a natural host species for ETX-mediated disease. First, this study showed that the prototoxin has a KEIS N-terminal sequence with a molecular mass of 33,054 Da. When the activation of ETX prototoxin ex vivo by goat small intestinal contents was assessed by SDS-PAGE, the prototoxin was processed in a stepwise fashion into an ~27-kDa band or higher-molecular-mass material that could be toxin oligomers. Purified ETX corresponding to the ~27-kDa band was cytotoxic. When it was biochemically characterized by mass spectrometry, the copresence of three ETX species, each with different C-terminal residues, was identified in the purified ~27-kDa ETX preparation. Cytotoxicity of each of the three ETX species was then demonstrated using recombinant DNA approaches. Serine protease inhibitors blocked the initial proteotoxin processing, while carboxypeptidase inhibitors blocked further processing events. Taken together, this study provides important new insights indicating that, in the intestinal lumen, serine protease (including trypsin and possibly chymotrypsin) initiates the processing of the prototoxin but other proteases, including carboxypeptidases, then process the prototoxin into multiple active and stable species. PMID:25336460

  10. Matriptase Proteolytically Activates Influenza Virus and Promotes Multicycle Replication in the Human Airway Epithelium

    PubMed Central

    Beaulieu, Alexandre; Gravel, Émilie; Cloutier, Alexandre; Marois, Isabelle; Colombo, Éloïc; Désilets, Antoine; Verreault, Catherine; Leduc, Richard; Marsault, Éric

    2013-01-01

    Influenza viruses do not encode any proteases and must rely on host proteases for the proteolytic activation of their surface hemagglutinin proteins in order to fuse with the infected host cells. Recent progress in the understanding of human proteases responsible for influenza virus hemagglutinin activation has led to the identification of members of the type II transmembrane serine proteases TMPRSS2 and TMPRSS4 and human airway trypsin-like protease; however, none has proved to be the sole enzyme responsible for hemagglutinin cleavage. In this study, we identify and characterize matriptase as an influenza virus-activating protease capable of supporting multicycle viral replication in the human respiratory epithelium. Using confocal microscopy, we found matriptase to colocalize with hemagglutinin at the apical surface of human epithelial cells and within endosomes, and we showed that the soluble form of the protease was able to specifically cleave hemagglutinins from H1 virus, but not from H2 and H3 viruses, in a broad pH range. We showed that small interfering RNA (siRNA) knockdown of matriptase in human bronchial epithelial cells significantly blocked influenza virus replication in these cells. Lastly, we provide a selective, slow, tight-binding inhibitor of matriptase that significantly reduces viral replication (by 1.5 log) of H1N1 influenza virus, including the 2009 pandemic virus. Our study establishes a three-pronged model for the action of matriptase: activation of incoming viruses in the extracellular space in its shed form, upon viral attachment or exit in its membrane-bound and/or shed forms at the apical surface of epithelial cells, and within endosomes by its membrane-bound form where viral fusion takes place. PMID:23365447

  11. Urinary proteolytic activation of renal epithelial Na+ channels in chronic heart failure

    PubMed Central

    Zheng, Hong; Liu, Xuefei; Sharma, Neeru M.; Li, Yulong; Pliquett, Rainer U; Patel, Kaushik P.

    2015-01-01

    One of the key mechanisms involved in renal Na+ retention in chronic heart failure (CHF) is activation of epithelial Na+ channels (ENaC) in collecting tubules. Proteolytic cleavage has an important role in activating ENaC. We hypothesized that enhanced levels of proteases in renal tubular fluid activate ENaC resulting in renal Na+ retention in rats with CHF. CHF was produced by left coronary artery ligation in rats. By immunoblotting, we found that several urinary serine proteases were significantly increased in CHF rats compared to sham rats (fold increases: furin 6.7, prostasin 23.6, plasminogen 2.06 and plasmin 3.57 vs. sham). Similar increases were observed in urinary samples from patients with CHF. Whole-cell patch-clamp was conducted in cultured renal collecting duct M-1 cells to record Na+ currents. Protease-rich urine (from rats and patients with CHF) significantly increased the Na+ inward current in M-1 cells. Two weeks of protease inhibitor treatment significantly abrogated the enhanced diuretic and natriuretic responses to ENaC inhibitor benzamil in rats with CHF. Increased podocyte lesions were observed in the kidneys of rats with CHF by transmission electron microscopy. Consistent with these results, podocyte damage markers desmin and podocin expressions were also increased in rats with CHF (increased ~2 folds). These findings suggest that podocyte damage may lead to increased proteases in the tubular fluid which in turn contributes to the enhanced renal ENaC activity, providing a novel mechanistic insight for Na+ retention commonly observed in CHF. PMID:26628676

  12. Liver and muscle proteolytic activity in field bean (Vicia faba L.) fed birds. Effect of vitamin E.

    PubMed

    Santidrián, S; Rodríguez, M L; Larralde, J

    1987-09-01

    Liver and muscle proteolytic activities (cathepsin A and D) were measured in growing male chickens fed ad libitum over periods of 30 and 60 days on 20% protein diets containing either heated soybean (HSB, control) or raw field bean (RFB, Vicia faba L.) as the main sources of protein. Vitamin E (250 mg/100 g diet) was added or not to the RFB diet. It has been found that in comparison to control HSB-fed animals, RFB-fed birds showed a significant reduction in the rate of growth, together with a significant increase in the activities of both cathepsins A and D in liver and muscle. The addition of vitamin E to the RFB diet had no significant effect on either weight gain or liver and muscle proteolytic activities. The possible nature of these effects is discussed.

  13. Healing activity of proteolytic fraction (P1G10) from Vasconcellea cundinamarcensis in a cutaneous wound excision model.

    PubMed

    Freitas, K M; Barcelos, L S; Caliari, M V; Salas, C E; Lopes, M T P

    2017-10-05

    The proteolytic enzymes from Vasconcellea cundinamarcensis have demonstrated efficacy to accelerate healing of skin lesions. We report here the efficacy of the proteolytic fraction - P1G10 during repair of excisional wounds in rodent model and analyze possible mediators involved. Using 0.05% P1G10 we observed on day 3rd increased wound contraction accompanied by an increase in activated neutrophils and VEGF relative to the control. On day 7th neutrophils returned to normal levels, and at 0.01% P1G10, an increase in NAG activity used to monitor monocyte/macrophage, was observed. On the other hand, on day 7th, we observed a decrease in TGF-β at 0.05% P1G10, accompanied by an increased transformation of the latent TGF-β to its active form. Also, on day 7th a reduction in MMP-9 activity and the number of apoptotic cells was observed along with an increase in fibroblast levels. Morphometrically, it appears that treatment with P1G10 accelerates the decline of initial inflammatory phase and reduces some unwanted effects likely caused by remaining TGF-β or MMPs, thus enhancing the quality of scar. Overall, these data suggest that the active proteolytic fraction P1G10 enhances the efficacy of repair in excisional cutaneous wounds. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  14. Antioxidants, free radicals, storage proteins, puroindolines, and proteolytic activities in bread wheat (Triticum aestivum) seeds during accelerated aging.

    PubMed

    Calucci, Lucia; Capocchi, Antonella; Galleschi, Luciano; Ghiringhelli, Silvia; Pinzino, Calogero; Saviozzi, Franco; Zandomeneghi, Maurizio

    2004-06-30

    Seeds of bread wheat were incubated at 40 degrees C and 100% relative humidity for 0, 3, 4, 6, and 10 days. The effects of accelerated aging on seed germinability and some biochemical properties of flour (carotenoid, free radical, and protein contents and proteolytic activity) and gluten (free radical content and flexibility) were investigated. Seed germinability decreased during aging, resulting in seed death after 10 days. A progressive decrease of carotenoid content, in particular, lutein, was observed, prolonging the incubation, whereas the free radical content increased in both flour and gluten. A degradation of soluble and storage proteins was found, associated with a marked increase of proteolytic activity and a loss of viscoelastic properties of gluten. On the contrary, puroindolines were quite resistant to the treatment. The results are discussed in comparison with those previously obtained during accelerated aging of durum wheat seeds.

  15. Identification of proteolytic activities in ROS 17/2.8 cell lysates which cleave peptide substrates for protein kinase C-mediated phosphorylation.

    PubMed

    Guidon, P T; Harrison, P

    1996-04-01

    We have observed two proteolytic activities in cell lysates from the rat osteoblastic osteosarcoma cell line ROS 17/2.8 which are capable of cleaving a peptide substrate for protein kinase C-mediated phosphorylation, and other peptides containing similar sequences. Both activities are inhibited by Pefabloc, a serine protease inhibitor, while one of the activities is inhibited by either EDTA or aprotinin. The protease inhibitors pepstatin, bestatin, E-64, leupeptin and phosphoramidon do not block either of these proteolytic activities.

  16. Proteolytic maturation of α2δ represents a checkpoint for activation and neuronal trafficking of latent calcium channels

    PubMed Central

    Kadurin, Ivan; Ferron, Laurent; Rothwell, Simon W; Meyer, James O; Douglas, Leon R; Bauer, Claudia S; Lana, Beatrice; Margas, Wojciech; Alexopoulos, Orpheas; Nieto-Rostro, Manuela; Pratt, Wendy S; Dolphin, Annette C

    2016-01-01

    The auxiliary α2δ subunits of voltage-gated calcium channels are extracellular membrane-associated proteins, which are post-translationally cleaved into disulfide-linked polypeptides α2 and δ. We now show, using α2δ constructs containing artificial cleavage sites, that this processing is an essential step permitting voltage-dependent activation of plasma membrane N-type (CaV2.2) calcium channels. Indeed, uncleaved α2δ inhibits native calcium currents in mammalian neurons. By inducing acute cell-surface proteolytic cleavage of α2δ, voltage-dependent activation of channels is promoted, independent from the trafficking role of α2δ. Uncleaved α2δ does not support trafficking of CaV2.2 channel complexes into neuronal processes, and inhibits Ca2+ entry into synaptic boutons, and we can reverse this by controlled intracellular proteolytic cleavage. We propose a model whereby uncleaved α2δ subunits maintain immature calcium channels in an inhibited state. Proteolytic processing of α2δ then permits voltage-dependent activation of the channels, acting as a checkpoint allowing trafficking only of mature calcium channel complexes into neuronal processes. DOI: http://dx.doi.org/10.7554/eLife.21143.001 PMID:27782881

  17. Metaproteomics of cellulose methanisation under thermophilic conditions reveals a surprisingly high proteolytic activity

    PubMed Central

    Lü, Fan; Bize, Ariane; Guillot, Alain; Monnet, Véronique; Madigou, Céline; Chapleur, Olivier; Mazéas, Laurent; He, Pinjing; Bouchez, Théodore

    2014-01-01

    Cellulose is the most abundant biopolymer on Earth. Optimising energy recovery from this renewable but recalcitrant material is a key issue. The metaproteome expressed by thermophilic communities during cellulose anaerobic digestion was investigated in microcosms. By multiplying the analytical replicates (65 protein fractions analysed by MS/MS) and relying solely on public protein databases, more than 500 non-redundant protein functions were identified. The taxonomic community structure as inferred from the metaproteomic data set was in good overall agreement with 16S rRNA gene tag pyrosequencing and fluorescent in situ hybridisation analyses. Numerous functions related to cellulose and hemicellulose hydrolysis and fermentation catalysed by bacteria related to Caldicellulosiruptor spp. and Clostridium thermocellum were retrieved, indicating their key role in the cellulose-degradation process and also suggesting their complementary action. Despite the abundance of acetate as a major fermentation product, key methanogenesis enzymes from the acetoclastic pathway were not detected. In contrast, enzymes from the hydrogenotrophic pathway affiliated to Methanothermobacter were almost exclusively identified for methanogenesis, suggesting a syntrophic acetate oxidation process coupled to hydrogenotrophic methanogenesis. Isotopic analyses confirmed the high dominance of the hydrogenotrophic methanogenesis. Very surprising was the identification of an abundant proteolytic activity from Coprothermobacter proteolyticus strains, probably acting as scavenger and/or predator performing proteolysis and fermentation. Metaproteomics thus appeared as an efficient tool to unravel and characterise metabolic networks as well as ecological interactions during methanisation bioprocesses. More generally, metaproteomics provides direct functional insights at a limited cost, and its attractiveness should increase in the future as sequence databases are growing exponentially. PMID:23949661

  18. Oxidative metabolism in guinea pig ventricular myocytes protected from proteolytic enzyme activity.

    PubMed

    Bailey, L E; Carlos, H; Amian, A; Moon, K E

    1987-07-01

    Surface structures on guinea pig ventricular myocytes were protected from proteolytic enzyme activity with 100 KIU.ml-2 aprotinin during mechanical disaggregation. Intact myocytes, approximately 7.5 X 10(6) cells.g-1 ventricular wet weight, were separated from debris and damaged cells using Cytodex I tissue culture supports. Cellular ultrastructure did not differ from that observed in intact tissue. Neither spontaneous contractions nor contracture were ever observed in these myocytes in calcium concentrations of 10 mmol.litre-1. Dinitrophenol (0.2 mmol. litre-1) uncoupled respiration in the myocytes but only after the sarcolemma had been disrupted with Triton X100. The adenosine diphosphate to oxygen ratio of mitochondria isolated from the myocytes was 2.4(0.2) and the respiratory control index 2.6(0.3). Calcium (1.8 mmol.litre-1) increased oxygen uptake in the presence of 10 mmol.litre-1 pyruvate or 11 mmol.litre-1 glucose but not 17 mmol. litre-1 succinate. Succinate dependent oxygen consumption was greater than pyruvate dependent oxygen consumption (1090.0(190.0) and 40.1(0.8) nl.min-1.mg-1 protein respectively). The Crabtree effect was present. Oxidative metabolism was normal in cells stored at 10 degrees C for seven days but deteriorated rapidly thereafter. The results indicate that myocytes disaggregated by this procedure retain many of the morphological and metabolic characteristics of intact cardiac muscle cells and are relatively homogeneous with respect to calcium tolerance and metabolic function.

  19. Proteolytic Enzymes in Detergents: Evidence of Their Presence through Activity Measurements Based on Electrophoresis

    ERIC Educational Resources Information Center

    Saperas, Nuria; Fonfria-Subiros, Elsa

    2011-01-01

    This laboratory exercise uses a problem-based approach to expose students to some basic concepts relating to proteins and enzymes. One of the main applications of enzymes at the industrial level is their use in the detergent market. The students examine a detergent sample to ascertain whether proteolytic enzymes are a component and, if so, which…

  20. Proteolytic Enzymes in Detergents: Evidence of Their Presence through Activity Measurements Based on Electrophoresis

    ERIC Educational Resources Information Center

    Saperas, Nuria; Fonfria-Subiros, Elsa

    2011-01-01

    This laboratory exercise uses a problem-based approach to expose students to some basic concepts relating to proteins and enzymes. One of the main applications of enzymes at the industrial level is their use in the detergent market. The students examine a detergent sample to ascertain whether proteolytic enzymes are a component and, if so, which…

  1. Circulating extracellular proteasome in the cerebrospinal fluid: a study on concentration and proteolytic activity.

    PubMed

    Mueller, Oliver; Anlasik, Timur; Wiedemann, Jonas; Thomassen, Jan; Wohlschlaeger, Jeremias; Hagel, Vincent; Keyvani, Kathy; Schwieger, Isabel; Dahlmann, Burkhardt; Sure, Ulrich; Sixt, Stephan Urs

    2012-03-01

    Alterations of the intracellular ubiquitin-proteasome pathway are found in neurodegenerative and inflammatory disorders of the central nervous system, as well as in its malignancies. Inhibitory substrates of the proteasomes represent promising approaches to control autoimmune inflammations and induction of apoptosis in cancer cells. Extracellular circulating proteasomes are positively correlated to outcome prognosis in hematogenic neoplasias and the outcome in critically ill patients. Previously, we reported raised levels of proteolytic active 20S proteasomes in the extracellular alveolar space in patients with acute respiratory distress syndrome (ARDS). For the cerebrospinal fluid, we assumed that extracellular circulating proteasomes with enzymatic activity can be found, too. Cerebrospinal fluid (CSF) samples of twenty-six patients (14 females, 12 males), who underwent diagnostic spinal myelography, were analyzed for leukocyte cell count, total protein content, lactate and interleukine-6 (Il-6) concentrations. CSF samples were analyzed for concentration and enzymatic activity of extracellular 20S proteasomes (fluorescenic substrate cleavage; femtokatal). Blood samples were analyzed with respect to concentration of extracellular circulating proteasomes. Choroidal plexus was harvested at autopsies and examined with immunoelectron microscopy (EM) for identification of possible transportation mechanisms. Statistical analysis was performed using SPSS (18.0.3). In all patients, extracellular proteasome was found in the CSF. The mean concentration was 24.6 ng/ml. Enzymatic activity of the 20S subunits of proteasomes was positively identified by the fluorescenic subtrate cleavage at a mean of 8.5 fkat/ml. Concentrations of extracellular proteasomes in the CSF, total protein content and Il-6 were uncorrelated. Immunoelectron microscopy revealed merging vesicles of proteasomes with the outer cell membrane suggestive of an exozytic transport mechanism. For the first time

  2. Luteinizing hormone stimulation of in vitro ovulation in brook trout (Salvelinus fontinalis) involves follicle contraction and activation of proteolytic genes.

    PubMed

    Crespo, Diego; Pramanick, Kousik; Goetz, Frederick W; Planas, Josep V

    2013-07-01

    Luteinizing hormone (LH) is an essential hormone for the stimulation of the ovulatory process in vertebrates. However, little is known in fish regarding the different mechanisms induced by LH during ovulation that facilitate the rupture of the follicle wall and the subsequent expulsion of the mature oocyte. In this study, the effects of salmon LH (sLH) on in vitro ovulation were investigated in brook trout (Salvelinus fontinalis) isolated follicles. sLH significantly stimulated in vitro ovulation and contraction of brook trout preovulatory follicles. In order to investigate the possible involvement of proteolytic events in the ovulatory action of LH, the expression of genes known to have a crucial role in the degradation of follicle wall structure was examined. Our results show that sLH clearly stimulated the mRNA expression levels of matrix metalloproteinases (MMPs; including mmp2 and mmp19) and other enzymes with proteolytic action during ovulation, such as a disintegrin and metalloproteinase with thrombospondin-like motifs 1 (adamts1) and plasminogen (plg), in brook trout preovulatory follicles. In addition, the expression of mmp2, adamts1 and plg increased in brook trout follicles during the progression of LH-induced ovulation. Interestingly, the expression of tissue inhibitor of matrix metalloproteinase 2 (timp2), a known regulator of MMP2 activity, paralleled that of mmp2, suggesting the existence of a controlled mechanism of MMP2 action. Therefore, the known increase in proteolytic activity during ovulation in fish could be the result of the stimulation of the expression of proteolytic enzymes by LH in preovulatory follicles. We propose that LH may stimulate ovulation in brook trout follicles by stimulating proteolysis of the follicle wall and by stimulating follicle contraction. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. Proteolytic Activation Transforms Heparin Cofactor II into a Host Defense Molecule

    PubMed Central

    Kalle, Martina; Papareddy, Praveen; Kasetty, Gopinath; Tollefsen, Douglas M.; Malmsten, Martin; Mörgelin, Matthias

    2013-01-01

    The abundant serine proteinase inhibitor heparin cofactor II (HCII) has been proposed to inhibit extravascular thrombin. However, the exact physiological role of this plasma protein remains enigmatic. In this study, we demonstrate a previously unknown role for HCII in host defense. Proteolytic cleavage of the molecule induced a conformational change, thereby inducing endotoxin-binding and antimicrobial properties. Analyses employing representative peptide epitopes mapped these effects to helices A and D. Mice deficient in HCII showed increased susceptibility to invasive infection by Pseudomonas aeruginosa, along with a significantly increased cytokine response. Correspondingly, decreased levels of HCII were observed in wild-type animals challenged with bacteria or endotoxin. In humans, proteolytically cleaved HCII forms were detected during wounding and in association with bacteria. Thus, the protease-induced uncovering of cryptic epitopes in HCII, which transforms the molecule into a host defense factor, represents a previously unknown regulatory mechanism in HCII biology and innate immunity. PMID:23656734

  4. Short communication: Characteristics of proteolytic activities of endo- and exopeptidases in alfalfa herbage and their implications for proteolysis in silage.

    PubMed

    Tao, L; Guo, X S; Zhou, H; Undersander, D J; Nandety, A

    2012-08-01

    The pH optimum and thermostability of both exopeptidases and endopeptidases were investigated in this study to elucidate the possible role of plant proteases in proteolysis during ensiling of alfalfa herbage. Proteolytic activities of 4 classes of endopeptidases (i.e., serine, metallo, aspartic, and cysteine peptidase) and 5 classes of exopeptidases (i.e., aminopeptidase, carboxypeptidase, dipeptidase, dipeptidyl-peptidase, and tripeptidyl-peptidase) were examined within pH values of 3 to 9, and within temperatures from 20 to 90°C. Serine and metalloproteases, the principal endopeptidases that hydrolyzed most of the protein to nonprotein nitrogen in alfalfa silage, had optimum activities at pH 4. Among the major exopeptidases contributing protein degradation in ensiled alfalfa, dipeptidase and tripeptidyl-peptidase had stable activities between pH 4 and 6, and carboxypeptidase activity was optimal at pH 5. The optimum temperature for most peptidase activities was 40°C. Proteolytic activities of both endo- and exopeptidases increased with the elevation of incubating temperature from 20 to 40°C. The pH value in well-preserved alfalfa silage is often above 4.0, and the temperatures in the ensiled mass range from 25 to 40°C. Therefore, high proteolytic activities between pH 4 and 6 and the temperature range of ensiled alfalfa suggest that plant peptidases play a role in hydrolyzing protein during prolonged storage. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  5. Nano-zymography Using Laser-Scanning Confocal Microscopy Unmasks Proteolytic Activity of Cell-Derived Microparticles

    PubMed Central

    Briens, Aurélien; Gauberti, Maxime; Parcq, Jérôme; Montaner, Joan; Vivien, Denis; Martinez de Lizarrondo, Sara

    2016-01-01

    Cell-derived microparticles (MPs) are nano-sized vesicles released by activated cells in the extracellular milieu. They act as vectors of biological activity by carrying membrane-anchored and cytoplasmic constituents of the parental cells. Although detection and characterization of cell-derived MPs may be of high diagnostic and prognostic values in a number of human diseases, reliable measurement of their size, number and biological activity still remains challenging using currently available methods. In the present study, we developed a protocol to directly image and functionally characterize MPs using high-resolution laser-scanning confocal microscopy. Once trapped on annexin-V coated micro-wells, we developed several assays using fluorescent reporters to measure their size, detect membrane antigens and evaluate proteolytic activity (nano-zymography). In particular, we demonstrated the applicability and specificity of this method to detect antigens and proteolytic activities of tissue-type plasminogen activator (tPA), urokinase and plasmin at the surface of engineered MPs from transfected cell-lines. Furthermore, we were able to identify a subset of tPA-bearing fibrinolytic MPs using plasma samples from a cohort of ischemic stroke patients who received thrombolytic therapy and in an experimental model of thrombin-induced ischemic stroke in mice. Overall, this method is promising for functional characterization of cell-derived MPs. PMID:27022410

  6. High-affinity prorenin binding to cardiac man-6-P/IGF-II receptors precedes proteolytic activation to renin.

    PubMed

    Saris, J J; Derkx, F H; De Bruin, R J; Dekkers, D H; Lamers, J M; Saxena, P R; Schalekamp, M A; Jan Danser, A H

    2001-04-01

    Mannose-6-phosphate (man-6-P)/insulin-like growth factor-II (man-6-P/IgF-II) receptors are involved in the activation of recombinant human prorenin by cardiomyocytes. To investigate the kinetics of this process, the nature of activation, the existence of other prorenin receptors, and binding of native prorenin, neonatal rat cardiomyocytes were incubated with recombinant, renal, or amniotic fluid prorenin with or without man-6-P. Intact and activated prorenin were measured in cell lysates with prosegment- and renin-specific antibodies, respectively. The dissociation constant (K(d)) and maximum number of binding sites (B(max)) for prorenin binding to man-6-P/IGF-II receptors were 0.6 +/- 0.1 nM and 3,840 +/- 510 receptors/myocyte, respectively. The capacity for prorenin internalization was greater than 10 times B(max). Levels of internalized intact prorenin decreased rapidly (half-life = 5 +/- 3 min) indicating proteolytic prosegment removal. Prorenin subdivision into man-6-P-free and man-6-P-containing fractions revealed that only the latter was bound. Cells also bound and activated renal but not amniotic fluid prorenin. We concluded that cardiomyocytes display high-affinity binding of renal but not extrarenal prorenin exclusively via man-6-P/IGF-II receptors. Binding precedes internalization and proteolytic activation to renin thereby supporting the concept of cardiac angiotensin formation by renal prorenin.

  7. Identification of proteolytic bacteria from the Arctic Chukchi Sea expedition cruise and characterization of cold-active proteases.

    PubMed

    Park, Ha Ju; Lee, Yung Mi; Kim, Sunghui; Wi, Ah Ram; Han, Se Jong; Kim, Han-Woo; Kim, Il-Chan; Yim, Joung Han; Kim, Dockyu

    2014-10-01

    Following collection of seawater samples during an Arctic Chukchi Sea expedition cruise of the Korean icebreaker Araon in 2012, a total of 15,696 bacteria were randomly isolated from Marine Broth 2216 agar plates. Of these, 2,526 (16%) showed proteolytic activity and were identified as mainly Alteromonas (31%), Staphylococcus (27%), and Pseudoalteromonas (14%). Among the proteolytic strains, seven were selected based on their significant ability to grow and produce a halo on skim milk plates at low temperatures (<5°C) owing to cold-active proteases. These strains were affiliated with the genus Pseudoalteromonas and were divided into three groups based on phylogenetic analysis of the 16S rRNA genes. Profiling cell membrane fatty acids confirmed the 16S rRNA-based differentiation and revealed the accordance between the two analyses. Seven genes for serine protease precursors were amplified from the corresponding strains, and based on sequence similarities, these genes were divided into three groups that were identical to those identified by the 16S rRNA phylogenetic analysis. Three protease genes from the representative strains of each group were composed of 2,127-2,130 bp, encoding 708-709 amino acids, and these genes yielded products with calculated molecular weights of approximately 72.3-72.8 kDa. Amino acid sequence analysis suggested that the precursors are members of the subtilase serine endo- and exo-peptidase clan and contain four domains (signal peptide, N-terminal prosequence, catalytic domain, and two pre-peptidase C-terminal domains). Upon expression in E. coli, each recombinant protease exhibited proteolytic activity on zymogram gels.

  8. Proteolytic activity of extracellular products from Arthrobotrys musiformis and their effect in vitro against Haemonchus contortus infective larvae

    PubMed Central

    Acevedo-Ramírez, Perla María del Carmen; Figueroa-Castillo, Juan Antonio; Ulloa-Arvizú, Raúl; Martínez-García, Luz Gisela; Guevara-Flores, Alberto; Rendón, Juan Luis; Valero-Coss, Rosa Ofelia; Mendoza-de Gives, Pedro; Quiroz-Romero, Héctor

    2015-01-01

    Arthrobotrys musiformis is a nematophagous fungus with potential for the biological control of Haemonchus contortus larvae. This study aimed to identify and demonstrate the proteolytic activity of extracellular products from A musiformis cultured in a liquid medium against H contortus infective larvae. A musiformis was cultured on a solid medium and further grown in a liquid medium, which was then processed through ion exchange and hydrophobic interaction chromatography. The proteolytic activity of the purified fraction was assayed with either gelatin or bovine serum albumin as substrate. Optimum proteolytic activity was observed at pH 8 and a temperature of 37°C. Results obtained with specific inhibitors suggest the enzyme belongs to the serine-dependent protease family. The purified fraction concentrate from A musiformis was tested against H contortus infective larvae. A time-dependent effect was observed with 77 per cent immobility after 48 hours incubation, with alteration of the sheath. It is concluded that A musiformis is a potential candidate for biological control because of its resistant structures and also because of its excretion of extracellular products such as proteases. The present study contributes to the identification of one of the in vitro mechanisms of action of Amusiformis, namely the extracellular production of proteases against H contortus infective larvae. More investigations should be undertaken into how these products could be used to decrease the nematode population in sheep flocks under field conditions, thereby improving animal health while simultaneously diminishing the human and environmental impact of chemical-based drugs. PMID:26392902

  9. Diversity of proteolytic microbes isolated from Antarctic freshwater lakes and characteristics of their cold-active proteases

    NASA Astrophysics Data System (ADS)

    Matsui, Mihoko; Kawamata, Akinori; Kosugi, Makiko; Imura, Satoshi; Kurosawa, Norio

    2017-09-01

    Despite being an extreme environment, the water temperature of freshwater lakes in Antarctica reaches 10 °C in summer, accelerating biological activity. In these environments, proteolytic microbial decomposers may play a large role in protein hydrolysis. We isolated 71 microbial strains showing proteolytic activity at 4 °C from three Antarctic freshwater lakes. They were classified as bacteria (63 isolates) and eukaryotes (8 isolates). The bacterial isolates were classified into the genera Flavobacterium (28 isolates), Pseudomonas (14 isolates), Arthrobacter (10 isolates), Psychrobacter (7 isolates), Cryobacterium (2 isolates), Hymenobacter (1 isolate), and Polaromonas (1 isolate). Five isolates of Flavobacterium and one of Hymenobacter seemed to belong to novel species. All eukaryotic isolates belonged to Glaciozyma antarctica, a psychrophilic yeast species originally isolated from the Weddell Sea near the Joinville Island, Antarctica. A half of representative strains were psychrophilic and did not grow at temperatures above 25 °C. The protease secreted by Pseudomonas prosekii strain ANS4-1 showed the highest activity among all proteases from representative isolates. The results of inhibitor tests indicated that nearly all the isolates secreted metalloproteases. Proteases from four representative isolates retained more than 30% maximal activity at 0 °C. These results expand our knowledge about microbial protein degradation in Antarctic freshwater lakes.

  10. Hemolytic and proteolytic activities of Aeromonas hydrophila and Aeromonas veronii biovar sobria in broth and salmon extract at different temperatures.

    PubMed

    González-Rodríguez, María-Nieves; Santos, Jesús A; Otero, Andrés; García-López, Maria-Luisa

    2004-02-01

    Expression of hemolytic and proteolytic activities throughout the growth cycle was investigated with two enterotoxic aeromonad strains assigned to the species Aeromonas hydrophila and Aeromonas veronii biovar sobria. Although growth kinetic data were dependent on strain, temperature, and substrate, maximum populations attained were higher than 9 log CFU/ml in aerated tryptone soya broth plus yeast extract (TSBYE) and salmon extract within the range 4 to 28 degrees C. For both strains in TSBYE, variable amounts of hemolytic activity were first detected at any temperature when aeromonad counts were over 9 log CFU/ml. Afterwards, this activity increased up to similar levels (109 to 112 hemolytic units per ml) without a significant increase in populations. Salmon extract supported hemolysin synthesis at 28 but not 4 degrees C. Proteolytic activity of the A. hydrophila strain was only expressed in salmon extract at 28 degrees C, whereas A. veronii biovar sobria did at 28 degrees C in both substrates and at 10 degrees C in TSBYE.

  11. Proteolytic modification of swelling-activated Cl- current in LNCaP prostate cancer epithelial cells.

    PubMed

    Vitko, Yulia V; Pogorelaya, Nelli H; Prevarskaya, Natalia; Skryma, Roman; Shuba, Yaroslav M

    2002-08-01

    The effects of intracellular application of trypsin on the Cl- current induced by hypotonic cell swelling (I(Cl,swell)) in human prostate cancer epithelial cells (LNCaP) was studied using the patch-clamp technique. In cells predialyzed with 1 mg/mL trypsin, I(Cl,swell)) developed and diminished in response to the application and withdrawal of hypotonic solution about three times faster than that in control cells. In trypsin-infused cells, I(Cl,swell)) also had about twofold higher current density and displayed considerably slowed voltage-dependent inactivation, which was quite pronounced in control cells at potentials above +60 mV. Trypsin-induced modification of I(Cl,swell)) could be prevented by coinfusion of 10 mg/mL soybean trypsin inhibitor, suggesting that proteolytic cleavage of essential intracellular structural domains of the I(Cl,swell))-carrying volume-regulated anion channel (VRAC) was responsible for this functional modification. The effect of trypsin was not dependent on the presence of intracellular ATP. We conclude that VRACs, similarly to voltage-gated Na+, K+, and Cl- channels, possess intracellular inactivation domain(s) subjected to proteolytic cleavage that may function in conformity with the classical "ball-and-chain" inactivation model.

  12. Atrial natriuretic peptide degradation by CPA47 cells - Evidence for a divalent cation-independent cell-surface proteolytic activity

    NASA Technical Reports Server (NTRS)

    Frost, S. J.; Chen, Y. M.; Whitson, P. A.

    1992-01-01

    Atrial natriuretic peptide (ANP) is rapidly cleared and degraded in vivo. Nonguanylate-cyclase receptors (C-ANPR) and a metalloproteinase, neutral endopeptidase (EC 3.4.24.11) (NEP 24.11), are thought to be responsible for its metabolism. We investigated the mechanisms of ANP degradation by an endothelial-derived cell line, CPA47. CPA47 cells degraded 88 percent of 125I-ANP after 1 h at 37 degrees C as determined by HPLC. Medium preconditioned by these cells degraded 41 percent of the 125I-ANP, and this activity was inhibited by a divalent cation chelator, EDTA. Furthermore, a cell-surface proteolytic activity degraded 125I-ANP in the presence of EDTA when receptor-mediated endocytosis was inhibited either by low temperature (4 degrees C) or by hyperosmolarity at 37 degrees C. The metalloproteinase, NEP 24.11, is unlikely to be the cell-surface peptidase because 125I-ANP is degraded by CPA47 cells at 4 degrees C in the presence of 5 mM EDTA. These data indicate that CPA47 cells can degrade ANP by a novel divalent cation-independent cell-surface proteolytic activity.

  13. Atrial natriuretic peptide degradation by CPA47 cells - Evidence for a divalent cation-independent cell-surface proteolytic activity

    NASA Technical Reports Server (NTRS)

    Frost, S. J.; Chen, Y. M.; Whitson, P. A.

    1992-01-01

    Atrial natriuretic peptide (ANP) is rapidly cleared and degraded in vivo. Nonguanylate-cyclase receptors (C-ANPR) and a metalloproteinase, neutral endopeptidase (EC 3.4.24.11) (NEP 24.11), are thought to be responsible for its metabolism. We investigated the mechanisms of ANP degradation by an endothelial-derived cell line, CPA47. CPA47 cells degraded 88 percent of 125I-ANP after 1 h at 37 degrees C as determined by HPLC. Medium preconditioned by these cells degraded 41 percent of the 125I-ANP, and this activity was inhibited by a divalent cation chelator, EDTA. Furthermore, a cell-surface proteolytic activity degraded 125I-ANP in the presence of EDTA when receptor-mediated endocytosis was inhibited either by low temperature (4 degrees C) or by hyperosmolarity at 37 degrees C. The metalloproteinase, NEP 24.11, is unlikely to be the cell-surface peptidase because 125I-ANP is degraded by CPA47 cells at 4 degrees C in the presence of 5 mM EDTA. These data indicate that CPA47 cells can degrade ANP by a novel divalent cation-independent cell-surface proteolytic activity.

  14. [Effect of Azospirillum lectins on the Activity of Proteolytic Enzymes and Their Inhibitors in Wheat Seedling Roots].

    PubMed

    Alen'kina, S A; Nikitina, V E

    2015-01-01

    The lectins of associative nitrogen-fixing strains Azospirillum brasilense Sp7 and Sp245 were shown to exerte a multidirectional effect on the activity of acidic (pH 3.5), neutral (6.8), and alkaline (pH 7.8) proteinases. The lectin of the epiphytic A. brasilense Sp7 decreased proteolytic activity at all pH values, whereas the lectin of the endophytic A. brasilense Sp245 activated neutral and alkaline proteinases, while not affecting the alkaline ones. Experiments with protease inhibitors made it possible to conclude that the lectins of the studied A. brasilense strains alter the ratio between the activities of different protease types in germinating seeds. The activity of trypsin inhibitors in wheat seedling roots was found to increase in the presence of the lectins. Our results indicate a broader spectrum of effects of azospirilla lectins on the host plant organism.

  15. Tuning Liposome Membrane Permeability by Competitive Peptide Dimerization and Partitioning-Folding Interactions Regulated by Proteolytic Activity.

    PubMed

    Lim, Seng Koon; Sandén, Camilla; Selegård, Robert; Liedberg, Bo; Aili, Daniel

    2016-02-19

    Membrane active peptides are of large interest for development of drug delivery vehicles and therapeutics for treatment of multiple drug resistant infections. Lack of specificity can be detrimental and finding routes to tune specificity and activity of membrane active peptides is vital for improving their therapeutic efficacy and minimize harmful side effects. We describe a de novo designed membrane active peptide that partition into lipid membranes only when specifically and covalently anchored to the membrane, resulting in pore-formation. Dimerization with a complementary peptide efficiently inhibits formation of pores. The effect can be regulated by proteolytic digestion of the inhibitory peptide by the matrix metalloproteinase MMP-7, an enzyme upregulated in many malignant tumors. This system thus provides a precise and specific route for tuning the permeability of lipid membranes and a novel strategy for development of recognition based membrane active peptides and indirect enzymatically controlled release of liposomal cargo.

  16. Tuning Liposome Membrane Permeability by Competitive Peptide Dimerization and Partitioning-Folding Interactions Regulated by Proteolytic Activity

    NASA Astrophysics Data System (ADS)

    Lim, Seng Koon; Sandén, Camilla; Selegård, Robert; Liedberg, Bo; Aili, Daniel

    2016-02-01

    Membrane active peptides are of large interest for development of drug delivery vehicles and therapeutics for treatment of multiple drug resistant infections. Lack of specificity can be detrimental and finding routes to tune specificity and activity of membrane active peptides is vital for improving their therapeutic efficacy and minimize harmful side effects. We describe a de novo designed membrane active peptide that partition into lipid membranes only when specifically and covalently anchored to the membrane, resulting in pore-formation. Dimerization with a complementary peptide efficiently inhibits formation of pores. The effect can be regulated by proteolytic digestion of the inhibitory peptide by the matrix metalloproteinase MMP-7, an enzyme upregulated in many malignant tumors. This system thus provides a precise and specific route for tuning the permeability of lipid membranes and a novel strategy for development of recognition based membrane active peptides and indirect enzymatically controlled release of liposomal cargo.

  17. Spatial characterization of proteolytic enzyme activity in the foregut region of the adult necrophagous fly, Protophormia terraenovae.

    PubMed

    Rivers, David B; Acca, Gillian; Fink, Marc; Brogan, Rebecca; Schoeffield, Andrew

    2014-08-01

    The spatial distribution of proteolytic enzymes in the adult foregut of Protophormia terraenovae was studied in the context of protein digestion and regurgitation. Based on substrate specificity, pH optima, and use of specific protease inhibitors, all adults tested displayed enzyme activity in the foregut consistent with pepsin, trypsin and chymotrypsin. Chymotrypsin-like and trypsin-like enzyme activity were detected in all gut fluids and tissues tested, with chymotrypsin displaying the highest activity in saliva and salivary gland tissue, whereas maximal trypsin activity was evident in the crop. Pepsin-like activity was only evident in crop fluids and tissues. The activity of all three enzymes was low or undetectable (pepsin) in the fluids and tissue homogenates derived from the esophagus and cardia of any of the adults assayed. Fed adult females displayed higher enzyme activities than fed males, and the activity of all three enzymes were much more prevalent in fed adults than starved. The pH optimum of the trypsin-like enzyme was between pH 7.0 and 8.0; chymotrypsin was near pH 8.0; and maximal pepsin-like activity occurred between pH 1.0 and 2.0. Regurgitate from fed adult females displayed enzyme activity consistent with the proteolytic enzymes detected in crop gut fluids. Enzymes in regurgitate were not derived from food sources based on assays of bovine liver samples. These latter observations suggest that adult flies release fluids from foregut when encountering dry foods, potentially as a means to initiate extra-oral digestion. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Proteolytic systems and AMP-activated protein kinase are critical targets of acute myeloid leukemia therapeutic approaches

    PubMed Central

    Pereira, Olga; Sampaio-Marques, Belém; Paiva, Artur; Correia-Neves, Margarida; Castro, Isabel; Ludovico, Paula

    2015-01-01

    The therapeutic strategies against acute myeloid leukemia (AML) have hardly been modified over four decades. Although resulting in a favorable outcome in young patients, older individuals, the most affected population, do not respond adequately to therapy. Intriguingly, the mechanisms responsible for AML cells chemoresistance/susceptibility are still elusive. Mounting evidence has shed light on the relevance of proteolytic systems (autophagy and ubiquitin-proteasome system, UPS), as well as the AMPK pathway, in AML biology and treatment, but their exact role is still controversial. Herein, two AML cell lines (HL-60 and KG-1) were exposed to conventional chemotherapeutic agents (cytarabine and/or doxorubicin) to assess the relevance of autophagy and UPS on AML cells’ response to antileukemia drugs. Our results clearly showed that the antileukemia agents target both proteolytic systems and the AMPK pathway. Doxorubicin enhanced UPS activity while drugs’ combination blocked autophagy specifically on HL-60 cells. In contrast, KG-1 cells responded in a more subtle manner to the drugs tested consistent with the higher UPS activity of these cells. In addition, the data demonstrates that autophagy may play a protective role depending on AML subtype. Specific modulators of autophagy and UPS are, therefore, promising targets for combining with standard therapeutic interventions in some AML subtypes. PMID:25537507

  19. The tissue plasminogen activator-plasminogen proteolytic cascade accelerates amyloid-beta (Abeta) degradation and inhibits Abeta-induced neurodegeneration.

    PubMed

    Melchor, Jerry P; Pawlak, Robert; Strickland, Sidney

    2003-10-01

    Accumulation of the amyloid-beta (Abeta) peptide depends on both its generation and clearance. To better define clearance pathways, we have evaluated the role of the tissue plasminogen activator (tPA)-plasmin system in Abeta degradation in vivo. In two different mouse models of Alzheimer's disease, chronically elevated Abeta peptide in the brain correlates with the upregulation of plasminogen activator inhibitor-1 (PAI-1) and inhibition of the tPA-plasmin system. In addition, Abeta injected into the hippocampus of mice lacking either tPA or plasminogen persists, inducing PAI-1 expression and causing activation of microglial cells and neuronal damage. Conversely, Abeta injected into wild-type mice is rapidly cleared and does not cause neuronal degeneration. Thus, the tPA-plasmin proteolytic cascade aids in the clearance of Abeta, and reduced activity of this system may contribute to the progression of Alzheimer's disease.

  20. A New Two-Component Regulatory System Involved in Adhesion, Autolysis, and Extracellular Proteolytic Activity of Staphylococcus aureus

    PubMed Central

    Fournier, Bénédicte; Hooper, David C.

    2000-01-01

    A transposition mutant of Staphylococcus aureus was selected from the parent strain MT23142, a derivative of strain 8325. The site of transposition was near the 5′ terminus of the gene arlS. ArlS exhibits strong similarities with histidine protein kinases. Sequence analysis suggested that arlS forms an operon with upstream gene arlR. The predicted product of arlR is a member of the OmpR-PhoB family of response regulators. The arlS mutant formed a biofilm on a polystyrene surface unlike the parent strain and the complemented mutant. Biofilm formation was associated with increased primary adherence to polystyrene, whereas cellular adhesion was only slightly decreased. In addition, the arlS mutant exhibited increased autolysis and altered peptidoglycan hydrolase activity compared to the parental strain and to the complemented mutant. As it has been shown for coagulase-negative staphylococci that some autolysins are able to bind polymer surfaces, these data suggest that the two-component regulatory system ArlS-ArlR may control attachment to polymer surfaces by affecting secreted peptidoglycan hydrolase activity. Finally, the arlS mutant showed a dramatic decrease of extracellular proteolytic activity, including serine protease activity, in comparison to the wild-type strain and the complemented mutant, and cells grown in the presence of phenylmethylsulfonyl fluoride (a serine protease inhibitor) showed an increased autolysin activity. Since the locus arlR-arlS strikingly modifies extracellular proteolytic activity, this locus might also be involved in the virulence of S. aureus. PMID:10869073

  1. Influence of lactic acid bacteria on redox status and on proteolytic activity of buckwheat (Fagopyrum esculentum Moench) sourdoughs.

    PubMed

    Capuani, Alessandro; Behr, Jürgen; Vogel, Rudi F

    2013-07-15

    Redox potential and proteolysis determine protein networks in doughs and thus dough rheology as well as the structure of baked goods. Namely, gluten-free bakery products needs structural improvements but little is known about these parameters in gluten free dough systems. In this work the influence of lactic acid bacteria (LAB) on redox status and proteolysis of buckwheat sourdoughs was investigated. An increase of free thiol groups was detected as redox potential was decreasing during fermentation. Thiol content at 8 h was higher in doughs fermented with strains with high reductive activity, such as Weissella (W.) cibaria in comparison to Pediococcus (P.) pentosaceus, which exhibited a lower reducing activity. At 24 h each fermentation showed a similar content of free thiol groups. Endogenous buckwheat proteases were characterized using various protease inhibitors in buckwheat doughs. Until pH3.1 a proteolysis increase was monitored in doughs. Employed LAB didn't show any detectable extracellular proteolytic activity. Flour proteases are thus responsible for protein breakdown, and this was demonstrated comparing free amino nitrogen (FAN) values and protein electrophoretic patterns of sourdough fermentations with chemical acidified (CA) doughs. FAN content at 24 h using P. pentosaceus, proteolytic comparative strain of Enterococcus faecalis, W. cibaria, mixed culture (containing P. pentosaceus and W. cibaria), CA and CA doughs containing glutathione (GSH) reached 45.9±1.3, 42.4±1.3, 40±1, 31±2, 29±2 and 17.8±3.9 mmol kg(-1) flour, respectively. Proteolysis was mainly influenced by pH and incubation time. The addition of GSH showed a decrease of proteolysis and of free amino acids. CA doughs showed a higher total free amino acids content than sourdough fermented with LAB indicating their metabolization. Fermentations with high FAN values exhibited lower band intensity (analyzed under reducing condition) in electrophoretic patterns. These results show that

  2. Analysis of Cathepsin and Furin Proteolytic Enzymes Involved in Viral Fusion Protein Activation in Cells of the Bat Reservoir Host

    PubMed Central

    El Najjar, Farah; Lampe, Levi; Baker, Michelle L.; Wang, Lin-Fa; Dutch, Rebecca Ellis

    2015-01-01

    Bats of different species play a major role in the emergence and transmission of highly pathogenic viruses including Ebola virus, SARS-like coronavirus and the henipaviruses. These viruses require proteolytic activation of surface envelope glycoproteins needed for entry, and cellular cathepsins have been shown to be involved in proteolysis of glycoproteins from these distinct virus families. Very little is currently known about the available proteases in bats. To determine whether the utilization of cathepsins by bat-borne viruses is related to the nature of proteases in their natural hosts, we examined proteolytic processing of several viral fusion proteins in cells derived from two fruit bat species, Pteropus alecto and Rousettus aegyptiacus. Our work shows that fruit bat cells have homologs of cathepsin and furin proteases capable of cleaving and activating both the cathepsin-dependent Hendra virus F and the furin-dependent parainfluenza virus 5 F proteins. Sequence analysis comparing Pteropus alecto furin and cathepsin L to proteases from other mammalian species showed a high degree of conservation; however significant amino acid variation occurs at the C-terminus of Pteropus alecto furin. Further analysis of furin-like proteases from fruit bats revealed that these proteases are catalytically active and resemble other mammalian furins in their response to a potent furin inhibitor. However, kinetic analysis suggests that differences may exist in the cellular localization of furin between different species. Collectively, these results indicate that the unusual role of cathepsin proteases in the life cycle of bat-borne viruses is not due to the lack of active furin-like proteases in these natural reservoir species; however, differences may exist between furin proteases present in fruit bats compared to furins in other mammalian species, and these differences may impact protease usage for viral glycoprotein processing. PMID:25706132

  3. Resistance to the Antibody-Drug Conjugate T-DM1 Is Based in a Reduction in Lysosomal Proteolytic Activity.

    PubMed

    Ríos-Luci, Carla; García-Alonso, Sara; Díaz-Rodríguez, Elena; Nadal-Serrano, Mercedes; Arribas, Joaquín; Ocaña, Alberto; Pandiella, Atanasio

    2017-09-01

    Trastuzumab-emtansine (T-DM1) is an antibody-drug conjugate (ADC) that was approved recently to treat HER2(+) breast cancers. Despite its impressive clinical efficacy in many patients, intrinsic and acquired resistance to T-DM1 has emerged as a challenge. To identify mechanisms of T-DM1 resistance, we isolated several resistant HER2(+) clones exhibiting stable drug refractoriness in vitro and in vivo Genomic comparisons showed substantial differences among three of the isolated clones, indicating several potential mechanisms of resistance to T-DM1. However, we observed no differences in HER2 levels and signaling among the resistant models and parental HER2(+) cells. Bioinformatics studies suggested that intracellular trafficking of T-DM1 could underlie resistance to T-DM1, and systematic analysis of the path followed by T-DM1 showed that the early steps in the internalization of the drug were unaltered. However, in some of the resistant clones, T-DM1 accumulated in lysosomes. In these clones, lysosomal pH was increased and the proteolytic activity of these organelles was deranged. These results were confirmed in T-DM1-resistant cells from patient-derived HER2(+) samples. We postulate that resistance to T-DM1 occurs through multiple mechanisms, one of which is impaired lysosomal proteolytic activity. Because other ADC may use the same internalization-degradation pathway to deliver active payloads, strategies aimed at restoring lysosomal functionality might overcome resistance to ADC-based therapies and improve their effectiveness. Cancer Res; 77(17); 4639-51. ©2017 AACR. ©2017 American Association for Cancer Research.

  4. Screening for antimicrobial and proteolytic activities of lactic acid bacteria isolated from cow, buffalo and goat milk and cheeses marketed in the southeast region of Brazil.

    PubMed

    Tulini, Fabricio L; Hymery, Nolwenn; Haertlé, Thomas; Le Blay, Gwenaelle; De Martinis, Elaine C P

    2016-02-01

    Lactic acid bacteria (LAB) can be isolated from different sources such as milk and cheese, and the lipolytic, proteolytic and glycolytic enzymes of LAB are important in cheese preservation and in flavour production. Moreover, LAB produce several antimicrobial compounds which make these bacteria interesting for food biopreservation. These characteristics stimulate the search of new strains with technological potential. From 156 milk and cheese samples from cow, buffalo and goat, 815 isolates were obtained on selective agars for LAB. Pure cultures were evaluated for antimicrobial activities by agar antagonism tests and for proteolytic activity on milk proteins by cultivation on agar plates. The most proteolytic isolates were also tested by cultivation in skim milk followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the fermented milk. Among the 815 tested isolates, three of them identified as Streptococcus uberis (strains FT86, FT126 and FT190) were bacteriocin producers, whereas four other ones identified as Weissella confusa FT424, W. hellenica FT476, Leuconostoc citreum FT671 and Lactobacillus plantarum FT723 showed high antifungal activity in preliminary assays. Complementary analyses showed that the most antifungal strain was L. plantarum FT723 that inhibited Penicillium expansum in modified MRS agar (De Man, Rogosa, Sharpe, without acetate) and fermented milk model, however no inhibition was observed against Yarrowia lipolytica. The proteolytic capacities of three highly proteolytic isolates identified as Enterococcus faecalis (strains FT132 and FT522) and Lactobacillus paracasei FT700 were confirmed by SDS-PAGE, as visualized by the digestion of caseins and whey proteins (β-lactoglobulin and α-lactalbumin). These results suggest potential applications of these isolates or their activities (proteolytic activity or production of antimicrobials) in dairy foods production.

  5. Proteolytic Activity and Production of γ-Aminobutyric Acid by Streptococcus thermophilus Cultivated in Microfiltered Pasteurized Milk.

    PubMed

    Brasca, Milena; Hogenboom, Johannes A; Morandi, Stefano; Rosi, Veronica; D'Incecco, Paolo; Silvetti, Tiziana; Pellegrino, Luisa

    2016-11-16

    A set of 191 strains of Streptococcus thermophilus were preliminarily screened for the presence of the genes codifying for cell envelope-associated proteinase (prtS) and for glutamate decarboxylase (gadB) responsible for γ-aminobutyric acid (GABA) production. The growth and proteolytic activity of the gadB-positive strains (9 presenting the prtS gene and 11 lacking it) were studied in microfiltered pasteurized milk. Degradation of both caseins (capillary electrophoresis) and soluble nitrogen fractions (HPLC) and changes in the profile of free amino acids (FAAs; ion-exchange chromatography) were evaluated at inoculation and after 6 and 24 h of incubation at 41 °C. None of the strains was capable of hydrolyzing caseins and β-lactoglobulin, and only two hydrolyzed part of α-lactalbumin, these proteins being present in their native states in pasteurized milk. Contrarily, most strains were able to hydrolyze peptones and peptides. For initial growth, most strains relied on the FAAs present in milk, whereas, after 6 h, prtS(+) strains released variable amounts of FAA. One prtS(+) strain expressed a PrtS(-) phenotype, and two prtS(-) strains showed a rather intense proteolytic activity. Only five strains (all prtS(+)) produced GABA, in variable quantities (up to 100 mg/L) and at different rates, depending on the acidification strength. Addition of glutamate did not induce production of GABA in nonproducing strains that, however, unexpectedly were shown to adopt the degradation of arginine into citrulline and ornithine as an alternative acid resistance system and likely as a source of ATP.

  6. Hypochlorous acid generated by neutrophils inactivates ADAMTS13: an oxidative mechanism for regulating ADAMTS13 proteolytic activity during inflammation.

    PubMed

    Wang, Yi; Chen, Junmei; Ling, Minhua; López, José A; Chung, Dominic W; Fu, Xiaoyun

    2015-01-16

    ADAMTS13 is a plasma metalloproteinase that cleaves large multimeric forms of von Willebrand factor (VWF) to smaller, less adhesive forms. ADAMTS13 activity is reduced in systemic inflammatory syndromes, but the cause is unknown. Here, we examined whether neutrophil-derived oxidants can regulate ADAMTS13 activity. We exposed ADAMTS13 to hypochlorous acid (HOCl), produced by a myeloperoxidase-H2O2-Cl(-) system, and determined its residual proteolytic activity using both a VWF A2 peptide substrate and multimeric plasma VWF. Treatment with 25 nm myeloperoxidase plus 50 μm H2O2 reduced ADAMTS13 activity by >85%. Using mass spectrometry, we demonstrated that Met(249), Met(331), and Met(496) in important functional domains of ADAMTS13 were oxidized to methionine sulfoxide in an HOCl concentration-dependent manner. The loss of enzyme activity correlated with the extent of oxidation of these residues. These Met residues were also oxidized in ADAMTS13 exposed to activated human neutrophils, accompanied by reduced enzyme activity. ADAMTS13 treated with either neutrophil elastase or plasmin was inhibited to a lesser extent, especially in the presence of plasma. These observations suggest that oxidation could be an important mechanism for ADAMTS13 inactivation during inflammation and contribute to the prothrombotic tendency associated with inflammation.

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

    PubMed

    Cruz, Laura Nogueira da; Alves, Eduardo; Leal, Mônica Teixeira; Juliano, Maria A; Rosenthal, Philip J; Juliano, Luiz; Garcia, Celia R S

    2011-03-01

    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. Ca(2+) 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 Ca(2+) in their activity is not fully understood. Here we investigated the role of Ca(2+) in protease modulation among rodent Plasmodium spp. Using fluorescence resonance energy transfer (FRET) peptides, we verified protease activity elicited by Ca(2+) from the endoplasmatic reticulum (ER) after stimulation with thapsigargin (a sarco/endoplasmatic reticulum Ca(2+)-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) Ca(2+) chelators were used to investigate the role played by Ca(2+) 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 Ca(2+) between species of Plasmodium. Copyright © 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

  8. Muscle wasting in a rat model of long-lasting sepsis results from the activation of lysosomal, Ca2+ -activated, and ubiquitin-proteasome proteolytic pathways.

    PubMed Central

    Voisin, L; Breuillé, D; Combaret, L; Pouyet, C; Taillandier, D; Aurousseau, E; Obled, C; Attaix, D

    1996-01-01

    We studied the alterations in skeletal muscle protein breakdown in long lasting sepsis using a rat model that reproduces a sustained and reversible catabolic state, as observed in humans. Rats were injected intravenously with live Escherichia coli; control rats were pair-fed to the intake of infected rats. Rats were studied in an acute septic phase (day 2 postinfection), in a chronic septic phase (day 6), and in a late septic phase (day 10). The importance of the lysosomal, Ca2+ -dependent, and ubiquitin-proteasome proteolytic processes was investigated using proteolytic inhibitors in incubated epitrochlearis muscles and by measuring mRNA levels for critical components of these pathways. Protein breakdown was elevated during the acute and chronic septic phases (when significant muscle wasting occurred) and returned to control values in the late septic phase (when wasting was stopped). A nonlysosomal and Ca2+ -independent process accounted for the enhanced proteolysis, and only mRNA levels for ubiquitin and subunits of the 20 S proteasome, the proteolytic core of the 26 S proteasome that degrades ubiquitin conjugates, paralleled the increased and decreased rates of proteolysis throughout. However, increased mRNA levels for the 14-kD ubiquitin conjugating enzyme E2, involved in substrate ubiquitylation, and for cathepsin B and m-calpain were observed in chronic sepsis. These data clearly support a major role for the ubiquitin-proteasome dependent proteolytic process during sepsis but also suggest that the activation of lysosomal and Ca2+ -dependent proteolysis may be important in the chronic phase. PMID:8601625

  9. Stepwise proteolytic activation of type I procollagen to collagen within the secretory pathway of tendon fibroblasts in situ.

    PubMed

    Canty-Laird, Elizabeth G; Lu, Yinhui; Kadler, Karl E

    2012-01-15

    Proteolytic cleavage of procollagen I to collagen I is essential for the formation of collagen fibrils in the extracellular matrix of vertebrate tissues. Procollagen is cleaved by the procollagen N- and C-proteinases, which remove the respective N- and C-propeptides from procollagen. Procollagen processing is initiated within the secretory pathway in tendon fibroblasts, which are adept in assembling an ordered extracellular matrix of collagen fibrils in vivo. It was thought that intracellular processing was restricted to the TGN (trans-Golgi network). In the present study, brefeldin A treatment of tendon explant cultures showed that N-proteinase activity is present in the resulting fused ER (endoplasmic reticulum)-Golgi compartment, but that C-proteinase activity is restricted to the TGN in embryonic chick tendon fibroblasts. In late embryonic and postnatal rat tail and postnatal mouse tail tendon, C-proteinase activity was detected in TGN and pre-TGN compartments. Preventing activation of the procollagen N- and C-proteinases with the furin inhibitor Dec-RVKR-CMK (decanoyl-Arg-Val-Lys-Arg-chloromethylketone) indicated that only a fraction of intracellular procollagen cleavage was mediated by newly activated proteinases. In conclusion, the N-propeptides are removed earlier in the secretory pathway than the C-propeptides. The removal of the C-propeptides in post-Golgi compartments most probably indicates preparation of collagen molecules for fibril formation at the cell-matrix interface.

  10. Proteolytic activation of proapoptotic kinase protein kinase Cδ by tumor necrosis factor α death receptor signaling in dopaminergic neurons during neuroinflammation

    PubMed Central

    2012-01-01

    Background The mechanisms of progressive dopaminergic neuronal loss in Parkinson’s disease (PD) remain poorly understood, largely due to the complex etiology and multifactorial nature of disease pathogenesis. Several lines of evidence from human studies and experimental models over the last decade have identified neuroinflammation as a potential pathophysiological mechanism contributing to disease progression. Tumor necrosis factor α (TNF) has recently emerged as the primary neuroinflammatory mediator that can elicit dopaminergic cell death in PD. However, the signaling pathways by which TNF mediates dopaminergic cell death have not been completely elucidated. Methods In this study we used a dopaminergic neuronal cell model and recombinant TNF to characterize intracellular signaling pathways activated during TNF-induced dopaminergic neurotoxicity. Etanercept and neutralizing antibodies to tumor necrosis factor receptor 1 (TNFR1) were used to block TNF signaling. We confirmed the results from our mechanistic studies in primary embryonic mesencephalic cultures and in vivo using the stereotaxic lipopolysaccharide (LPS) model of nigral dopaminergic degeneration. Results TNF signaling in dopaminergic neuronal cells triggered the activation of protein kinase Cδ (PKCδ), an isoform of the novel PKC family, by caspase-3 and caspase-8 dependent proteolytic cleavage. Both TNFR1 neutralizing antibodies and the soluble TNF receptor Etanercept blocked TNF-induced PKCδ proteolytic activation. Proteolytic activation of PKCδ was accompanied by translocation of the kinase to the nucleus. Notably, inhibition of PKCδ signaling by small interfering (si)RNA or overexpression of a PKCδ cleavage-resistant mutant protected against TNF-induced dopaminergic neuronal cell death. Further, primary dopaminergic neurons obtained from PKCδ knockout (−/−) mice were resistant to TNF toxicity. The proteolytic activation of PKCδ in the mouse substantia nigra in the neuroinflammatory LPS

  11. Proteolytic activation of the SARS-coronavirus spike protein: cutting enzymes at the cutting edge of antiviral research.

    PubMed

    Simmons, Graham; Zmora, Pawel; Gierer, Stefanie; Heurich, Adeline; Pöhlmann, Stefan

    2013-12-01

    The severe acute respiratory syndrome (SARS) pandemic revealed that zoonotic transmission of animal coronaviruses (CoV) to humans poses a significant threat to public health and warrants surveillance and the development of countermeasures. The activity of host cell proteases, which cleave and activate the SARS-CoV spike (S) protein, is essential for viral infectivity and constitutes a target for intervention. However, the identities of the proteases involved have been unclear. Pioneer studies identified cathepsins and type II transmembrane serine proteases as cellular activators of SARS-CoV and demonstrated that several emerging viruses might exploit these enzymes to promote their spread. Here, we will review the proteolytic systems hijacked by SARS-CoV for S protein activation, we will discuss their contribution to viral spread in the host and we will outline antiviral strategies targeting these enzymes. This paper forms part of a series of invited articles in Antiviral Research on "From SARS to MERS: 10years of research on highly pathogenic human coronaviruses.'' Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Proteolytic activation of the SARS-coronavirus spike protein: Cutting enzymes at the cutting edge of antiviral research

    PubMed Central

    Simmons, Graham; Zmora, Pawel; Gierer, Stefanie; Heurich, Adeline; Pöhlmann, Stefan

    2013-01-01

    The severe acute respiratory syndrome (SARS) pandemic revealed that zoonotic transmission of animal coronaviruses (CoV) to humans poses a significant threat to public health and warrants surveillance and the development of countermeasures. The activity of host cell proteases, which cleave and activate the SARS-CoV spike (S) protein, is essential for viral infectivity and constitutes a target for intervention. However, the identities of the proteases involved have been unclear. Pioneer studies identified cathepsins and type II transmembrane serine proteases as cellular activators of SARS-CoV and demonstrated that several emerging viruses might exploit these enzymes to promote their spread. Here, we will review the proteolytic systems hijacked by SARS-CoV for S protein activation, we will discuss their contribution to viral spread in the host and we will outline antiviral strategies targeting these enzymes. This paper forms part of a series of invited articles in Antiviral Research on “From SARS to MERS: 10 years of research on highly pathogenic human coronaviruses.” PMID:24121034

  13. Influence of zinc on bacterial populations and their proteolytic enzyme activities in freshwater environments: a cross-site comparison.

    PubMed

    Rasmussen, Lauren; Olapade, Ola A

    2016-04-01

    Temporal responses of indigenous bacterial populations and proteolytic enzyme (i.e., aminopeptidase) activities in the bacterioplankton assemblages from 3 separate freshwater environments were examined after exposure to various zinc (Zn) concentrations under controlled microcosm conditions. Zn concentrations (ranging from 0 to 10 μmol/L) were added to water samples collected from the Kalamazoo River, Rice Creek, and Huron River and examined for bacterial abundance and aminopeptidase activities at various time intervals over a 48 h incubation period in the dark. The results showed that the Zn concentrations did not significantly influence total bacterial counts directly; however, aminopeptidase activities varied significantly to increasing zinc treatments over time. Also, analysis of variance and linear regression analyses revealed significant positive relationships between bacterial numbers and their hydrolytic enzyme activities, suggesting that both probably co-vary with increasing Zn concentrations in aquatic systems. The results from this study serve as additional evidence of the ecological role of Zn as an extracellular peptidase cofactor on the dynamics of bacterial assemblages in aquatic environments.

  14. Proteolytic activities of kiwifruit actinidin (Actinidia deliciosa cv. Hayward) on different fibrous and globular proteins: a comparative study of actinidin with papain.

    PubMed

    Chalabi, Maryam; Khademi, Fatemeh; Yarani, Reza; Mostafaie, Ali

    2014-04-01

    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.

  15. Digestive proteolytic and amylolytic activities and feeding responses of Helicoverpa armigera (Lepidoptera: Noctuidae) on different host plants.

    PubMed

    Hemati, S A; Naseri, B; Ganbalani, G Nouri; Dastjerdi, H Rafiee; Golizadeh, A

    2012-08-01

    Digestive proteolytic and amylolytic activities and feeding responses of fifth instar larvae of Helicoverpa armigera (Hübner) on different host plants including chickpea (cultivars Arman, Hashem, Azad, and Binivich), common bean (cultivar Khomein), white kidney bean (cultivar Dehghan), red kidney bean (cultivar Goli), cowpea (cultivar Mashhad), tomato (cultivar Meshkin), and potato (cultivars Agria and Satina) were studied under laboratory conditions (25 +/- 1 degrees C, 65 +/- 5% RH and a photoperiod of 16:8 [L:D] h). Our results showed that the highest protease activity in optimal pH was on cultivar Dehghan (8.717 U/mg) and lowest one was on Meshkin (3.338 U/mg). In addition, the highest amylase activity in optimal pH was on cultivar Dehghan (0.340 mU/mg) and lowest was on Meshkin (0.088 mU/mg). The larval weight of fifth instar H. armigera showed significant difference, being heaviest on Binivich (125.290 +/- 5.050 mg) and lightest on Meshkin (22.773 +/- 0.575 mg). Furthermore, the highest and lowest values of food consumed were on Goli (362.800 +/- 27.500 mg) and Satina (51.280 +/- 4.500 mg), respectively. In addition, the lowest values of prepupal and pupal weight were on Meshkin (32.413 +/- 0.980 and 41.820 +/- 1.270 mg, respectively). The results indicated that tomato (Meshkin) was unsuitable host for feeding fifth instar larvae of H. armigera.

  16. Condurango glycoside-rich components stimulate DNA damage-induced cell cycle arrest and ROS-mediated caspase-3 dependent apoptosis through inhibition of cell-proliferation in lung cancer, in vitro and in vivo.

    PubMed

    Sikdar, Sourav; Mukherjee, Avinaba; Ghosh, Samrat; Khuda-Bukhsh, Anisur Rahman

    2014-01-01

    Chemotherapeutic potential of Condurango glycoside-rich components (CGS) was evaluated in NSCLC, in vitro and in BaP-intoxicated rats, in vivo. NSCLC cells were treated with different concentrations of CGS to test their effect on cell viability. Cellular morphology, DNA-damage, AnnexinV-FITC/PI, cell cycle regulation, ROS-accumulation, MMP, and expressions of related signalling genes were critically analysed. 0.22 μg/μl CGS (IC₅₀ dose at 24 h) was selected for the study. CGS-induced apoptosis via DNA damage was evidenced by DNA-ladder formation, increase of AnnexinV-positive cells, cell cycle arrest at subG0/G1 and differential expressions of apoptotic genes. ROS-elevation and MMP-depolarization with significant caspase-3 activation might lead to apoptotic cell death. Anti-proliferative activity was confirmed by EGFR-expression modulation. ROS accumulation and DNA-nick formation with tissue damage-repair activity after post-cancerous CGS treatment, in vivo, supported the in vitro findings. Overall results advocate considerable apoptosis-inducing potential of CGS against NSCLC, validating its use against lung cancer by CAM practitioners. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. Proteolytic activity is altered in brain tissue of rats upon chronic exposure to ozone

    SciTech Connect

    Benuck, M.; Banay-Schwartz, M.; Lajtha, A. )

    1993-01-01

    Tissue from pons medulla of rats exposed in vivo to various levels of ozone was assayed for calpain and cathepsin D activity. Chronic exposure to ozone increased calpain activity, which was 35% to 46% higher in the homogenates of animals exposed to 1.0 ppm ozone than in those of animals exposed to 0.5 ppm ozone or of controls. An increase in activity of 26% was also observed in the soluble supernatant. The increase in activity did not seem to be caused by ozone effects on calpastatin. Addition of 32 mM carnitine to the incubation mixture increased total activity 3-4 fold, making the differences in activity proportionately smaller. Cathepsin D activity was little altered. Changes in calpain activity and in the generation of free oxygen radicals have been implicated in the aging process, long-term exposure to ozone may magnify changes. Ozone exposure may cause changes in brain protein metabolism. 15 refs., 2 tabs.

  18. Procyanidin-rich extract of natural cocoa powder causes ROS-mediated caspase-3 dependent apoptosis and reduction of pro-MMP-2 in epithelial ovarian carcinoma cell lines.

    PubMed

    Taparia, Shruti Sanjay; Khanna, Aparna

    2016-10-01

    Over the last four centuries, cocoa and chocolate have been described as having potential medicinal value. As of today, Theobroma cacao L. (Sterculiaceae) and its products are consumed worldwide. They are of great research interest because of the concentration dependent antioxidant as well as pro-oxidant properties of some of their polyphenolic constituents, specially procyanidins and flavan-3-ols such as catechin. This study was aimed at investigating the cellular and molecular changes associated with cytotoxicity, caused due pro-oxidant activity of cocoa catechins and procyanidins, in ovarian cancer cell lines. Extract of non-alkalized cocoa powder enriched with catechins and procyanidins was used to treat human epithelial ovarian cancer cell lines OAW42 and OVCAR3 at various concentrations ≤1000μg/mL. The effect of treatment on intracellular reactive oxygen species (ROS) levels was determined. Apoptotic cell death, post treatment, was evaluated microscopically and using flow cytometry by means of annexin-propidium iodide (PI) dual staining. Levels of active caspase-3 as a pro-apoptotic marker and matrix metalloproteinase 2 (MMP2) as an invasive potential marker were detected using Western blotting and gelatin zymography. Treatment with extract caused an increase in intracellular ROS levels in OAW42 and OVCAR3 cell lines. Bright field and fluorescence microscopy of treated cells revealed apoptotic morphology and DNA damage. Increase in annexin positive cell population and dose dependent upregulation of caspase-3 confirmed apoptotic cell death. pro-MMP2 was found to be downregulated in a dose dependent manner in cells treated with the extract. Treated cells also showed a reduction in MMP2 activity. Our data suggests that cocoa catechins and procyanidins are cytotoxic to epithelial ovarian cancer, inducing apoptotic morphological changes, DNA damage and caspase-3 mediated cell death. Downregulation of pro-MMP2 and reduction in active MMP2 levels imply a decrease

  19. FRETS-VWF73 rather than CBA assay reflects ADAMTS13 proteolytic activity in acquired thrombotic thrombocytopenic purpura patients.

    PubMed

    Mancini, I; Valsecchi, C; Lotta, L A; Deforche, L; Pontiggia, S; Bajetta, M; Palla, R; Vanhoorelbeke, K; Peyvandi, F

    2014-08-01

    Collagen-binding activity (CBA) and FRETS-VWF73 assays are widely adopted methods for the measurement of the plasmatic activity of ADAMTS13, the von Willebrand factor (VWF) cleaving-protease. Accurately assessing the severe deficiency of ADAMTS13 is important in the management of thrombotic thrombocytopenic purpura (TTP). However, non-concordant results between the two assays have been reported in a small but relevant percentage of TTP cases. We investigated whether CBA or FRETS-VWF73 assay reflects ADAMTS13 proteolytic activity in acquired TTP patients with non-concordant measurements. Twenty plasma samples with non-concordant ADAMTS13 activity results, <10% using FRETS-VWF73 and ≥20% using CBA, and 11 samples with concordant results, <10% using either FRETS-VWF73 and CBA assays, were analysed. FRETS-VWF73 was performed in the presence of 1.5 M urea. ADAMTS13 activities were also measured under flow conditions and the VWF multimer pattern was defined in order to verify the presence of ultra-large VWF due to ADAMTS13 deficiency. In FRETS-VWF73 assay with 1.5 M urea, ADAMTS13 activity significantly increased in roughly 50% of the samples with non-concordant results, whereas it remained undetectable in all samples with concordant measurements. Under flow conditions, all tested samples showed reduced ADAMTS13 activity. Finally, samples with non-concordant results showed a ratio of high molecular weight VWF multimers higher than normal. Our results support the use of FRETS-VWF73 over CBA assay for the assessment of ADAMTS13 severe deficiency and indicate urea as one cause of the observed differences.

  20. Autophagic Signaling and Proteolytic Enzyme Activity in Cardiac and Skeletal Muscle of Spontaneously Hypertensive Rats following Chronic Aerobic Exercise

    PubMed Central

    McMillan, Elliott M.; Paré, Marie-France; Baechler, Brittany L.; Graham, Drew A.; Rush, James W. E.; Quadrilatero, Joe

    2015-01-01

    Hypertension is a cardiovascular disease associated with deleterious effects in skeletal and cardiac muscle. Autophagy is a degradative process essential to muscle health. Acute exercise can alter autophagic signaling. Therefore, we aimed to characterize the effects of chronic endurance exercise on autophagy in skeletal and cardiac muscle of normotensive and hypertensive rats. Male Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) were assigned to a sedentary condition or 6 weeks of treadmill running. White gastrocnemius (WG) of hypertensive rats had higher (p<0.05) caspase-3 and proteasome activity, as well as elevated calpain activity. In addition, skeletal muscle of hypertensive animals had elevated (p<0.05) ATG7 and LC3I protein, LAMP2 mRNA, and cathepsin activity, indicative of enhanced autophagic signaling. Interestingly, chronic exercise training increased (p<0.05) Beclin-1, LC3, and p62 mRNA as well as proteasome activity, but reduced (p<0.05) Beclin-1 and ATG7 protein, as well as decreased (p<0.05) caspase-3, calpain, and cathepsin activity. Left ventricle (LV) of hypertensive rats had reduced (p<0.05) AMPKα and LC3II protein, as well as elevated (p<0.05) p-AKT, p-p70S6K, LC3I and p62 protein, which collectively suggest reduced autophagic signaling. Exercise training had little effect on autophagy-related signaling factors in LV; however, exercise training increased (p<0.05) proteasome activity but reduced (p<0.05) caspase-3 and calpain activity. Our results suggest that autophagic signaling is altered in skeletal and cardiac muscle of hypertensive animals. Regular aerobic exercise can effectively alter the proteolytic environment in both cardiac and skeletal muscle, as well as influence several autophagy-related factors in skeletal muscle of normotensive and hypertensive rats. PMID:25799101

  1. Autophagic signaling and proteolytic enzyme activity in cardiac and skeletal muscle of spontaneously hypertensive rats following chronic aerobic exercise.

    PubMed

    McMillan, Elliott M; Paré, Marie-France; Baechler, Brittany L; Graham, Drew A; Rush, James W E; Quadrilatero, Joe

    2015-01-01

    Hypertension is a cardiovascular disease associated with deleterious effects in skeletal and cardiac muscle. Autophagy is a degradative process essential to muscle health. Acute exercise can alter autophagic signaling. Therefore, we aimed to characterize the effects of chronic endurance exercise on autophagy in skeletal and cardiac muscle of normotensive and hypertensive rats. Male Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) were assigned to a sedentary condition or 6 weeks of treadmill running. White gastrocnemius (WG) of hypertensive rats had higher (p<0.05) caspase-3 and proteasome activity, as well as elevated calpain activity. In addition, skeletal muscle of hypertensive animals had elevated (p<0.05) ATG7 and LC3I protein, LAMP2 mRNA, and cathepsin activity, indicative of enhanced autophagic signaling. Interestingly, chronic exercise training increased (p<0.05) Beclin-1, LC3, and p62 mRNA as well as proteasome activity, but reduced (p<0.05) Beclin-1 and ATG7 protein, as well as decreased (p<0.05) caspase-3, calpain, and cathepsin activity. Left ventricle (LV) of hypertensive rats had reduced (p<0.05) AMPKα and LC3II protein, as well as elevated (p<0.05) p-AKT, p-p70S6K, LC3I and p62 protein, which collectively suggest reduced autophagic signaling. Exercise training had little effect on autophagy-related signaling factors in LV; however, exercise training increased (p<0.05) proteasome activity but reduced (p<0.05) caspase-3 and calpain activity. Our results suggest that autophagic signaling is altered in skeletal and cardiac muscle of hypertensive animals. Regular aerobic exercise can effectively alter the proteolytic environment in both cardiac and skeletal muscle, as well as influence several autophagy-related factors in skeletal muscle of normotensive and hypertensive rats.

  2. Proteolytic Activation of the Essential Parasitophorous Vacuole Cysteine Protease SERA6 Accompanies Malaria Parasite Egress from Its Host Erythrocyte*

    PubMed Central

    Ruecker, Andrea; Shea, Michael; Hackett, Fiona; Suarez, Catherine; Hirst, Elizabeth M. A.; Milutinovic, Katarina; Withers-Martinez, Chrislaine; Blackman, Michael J.

    2012-01-01

    The malaria parasite replicates within an intraerythrocytic parasitophorous vacuole (PV). The PV and host cell membranes eventually rupture, releasing merozoites in a process called egress. Certain inhibitors of serine and cysteine proteases block egress, indicating a crucial role for proteases. The Plasmodium falciparum genome encodes nine serine-repeat antigens (SERAs), each of which contains a central domain homologous to the papain-like (clan CA, family C1) protease family. SERA5 and SERA6 are indispensable in blood-stage parasites, but the function of neither is known. Here we show that SERA6 localizes to the PV where it is precisely cleaved just prior to egress by an essential serine protease called PfSUB1. Mutations that replace the predicted catalytic Cys of SERA6, or that block SERA6 processing by PfSUB1, could not be stably introduced into the parasite genomic sera6 locus, indicating that SERA6 is an essential enzyme and that processing is important for its function. We demonstrate that cleavage of SERA6 by PfSUB1 converts it to an active cysteine protease. Our observations reveal a proteolytic activation step in the malarial PV that may be required for release of the parasite from its host erythrocyte. PMID:22984267

  3. Proteolytic activity of Saccharomyces cerevisiae strains associated with Italian dry-fermented sausages in a model system.

    PubMed

    Chaves-López, Clemencia; Paparella, Antonello; Tofalo, Rosanna; Suzzi, Giovanna

    2011-10-17

    Strains of Saccharomyces cerevisiae isolated from Italian salami were screened for proteolytic activity in a model system containing sarcoplasmic (SMS) or myofibrillar (MMS) proteins, at 20°C for 14days, to evaluate the possible influence on the proteolysis of fermented sausages. SDS-PAGE revealed that 14 of the most osmotolerant strains were responsible for the extensive hydrolysis of the main myofibrillar proteins, while only one strain was able to hydrolyze sarcoplasmic proteins. Free amino acids (FAA) accumulated during proteolysis were strain-dependent with different patterns from sarcoplasmic or myofibrillar protein fraction. In general, proteolysis lead Cys, Glu, Lys and Val as the most abundant FAA in the inoculated MMS samples. Volatile compound analysis, determined by SPME-GC-MS, evidenced 3-methyl butanol in MMS, and 2-methyl propanol and 3-methyl-1-butanol in SMS as major compounds. Our findings highlight that S. cerevisiae could influence the composition in amino acids and volatile compounds in fermented sausages, with a strain-dependent activity.

  4. Structural basis for the ATP-independent proteolytic activity of LonB proteases and reclassification of their AAA+ modules.

    PubMed

    An, Young Jun; Na, Jung-Hyun; Kim, Myung-Il; Cha, Sun-Shin

    2015-10-01

    Lon proteases degrade defective or denature proteins as well as some folded proteins for the control of cellular protein quality. There are two types of Lon proteases, LonA and LonB. Each consists of two functional components: a protease component and an ATPase associated with various cellular activities (AAA+ module). Here, we report the 2.03 -resolution crystal structure of the isolated AAA+ module (iAAA+ module) of LonB from Thermococcus onnurineus NA1 (TonLonB). The iAAA+ module, having no bound nucleotide, adopts a conformation virtually identical to the ADP-bound conformation of AAA+ modules in the hexameric structure of TonLonB; this provides insights into the ATP-independent proteolytic activity observed in a LonB protease. Structural comparison of AAA+ modules between LonA and LonB revealed that the AAA+ modules of Lon proteases are separated into two distinct clades depending on their structural features. The AAA+ module of LonB belongs to the -H2 & Ins1 insert clade (HINS clade)- defined for the first time in this study, while the AAA+ module of LonA is a member of the HCLR clade.

  5. Structure-Based Design of Mucor pusillus Pepsin for the Improved Ratio of Clotting Activity/Proteolytic Activity in Cheese Manufacture.

    PubMed

    Zhang, Jie; Sun, Yonghai; Li, Zhuolin; Luo, Quan; Li, Tiezhu; Wang, Tuoyi

    2015-01-01

    Previous theoretical studies have determined the intermolecular interactions between Mucor pusillus pepsin (MPP) and the key domain of κ-casein, with the aim to understand the mechanism of milk clotting in the specific hydrolysis of κ-casein by MPP for cheese making. Here, we combined the docking model with site-directed mutagenesis to further investigate the functional roles of amino acid residues in the active site of MPP. T218S replacement caused a low thermostability and moderate increase in the clotting activity. Mutations of three amino acid residues, T218A and T218S in S2 region and L287G in S4 region, led to a significant decrease in proteolytic activity. For T218S and L287G, an increase in the ratio of clotting activity to proteolytic activity (C/P) was observed, in particular 3.34-fold increase was found for T218S mutants. Structural analysis of the binding mode of MPP and chymosin splitting domain (CSD) of κ-casein indicated that T218S plays a critical role in forming a hydrogen bond with the hydroxyl group of Ser(104) around the MPP-sensitive Phe(105)-Met(106) peptide bond of κ- casein and L287G is partially responsible for CSD accommodation in a suitable hydrophobic environment. These data suggested that T218S mutant could serve as a promising milk coagulant that contributes to an optimal flavor development in mature cheese.

  6. Decreased proteolytic activity of the mitochondrial amyloid-β degrading enzyme, PreP peptidasome, in Alzheimer's disease brain mitochondria.

    PubMed

    Alikhani, Nyosha; Guo, Lan; Yan, Shiqiang; Du, Heng; Pinho, Catarina Moreira; Chen, John Xi; Glaser, Elzbieta; Yan, Shirley ShiDu

    2011-01-01

    Accumulation of amyloid-β peptide (Aβ), the neurotoxic peptide implicated in the pathogenesis of Alzheimer's disease (AD), has been shown in brain mitochondria of AD patients and of AD transgenic mouse models. The presence of Aβ in mitochondria leads to free radical generation and neuronal stress. Recently, we identified the presequence protease, PreP, localized in the mitochondrial matrix in mammalian mitochondria as the novel mitochondrial Aβ-degrading enzyme. In the present study, we examined PreP activity in the mitochondrial matrix of the human brain's temporal lobe, an area of the brain highly susceptible to Aβ accumulation and reactive oxygen species (ROS) production. We found significantly lower hPreP activity in AD brains compared with non-AD age-matched controls. By contrast, in the cerebellum, a brain region typically spared from Aβ accumulation, there was no significant difference in hPreP activity when comparing AD samples to non-AD controls. We also found significantly reduced PreP activity in the mitochondrial matrix of AD transgenic mouse brains (Tg mAβPP and Tg mAβPP/ABAD) when compared to non-transgenic aged-matched mice. Furthermore, mitochondrial fractions isolated from AD brains and Tg mAβPP mice had higher levels of 4-hydroxynonenal, an oxidative product, as compared with those from non-AD and nonTg mice. Accordingly, activity of cytochrome c oxidase was significantly reduced in the AD mitochondria. These findings suggest that decreased PreP proteolytic activity, possibly due to enhanced ROS production, contributes to Aβ accumulation in mitochondria leading to the mitochondrial toxicity and neuronal death that is exacerbated in AD. Clearance of mitochondrial Aβ by PreP may thus be of importance in the pathology of AD.

  7. Characterisation of general proteolytic, milk clotting and antifungal activity of Ficus carica latex during fruit ripening.

    PubMed

    Raskovic, Brankica; Lazic, Jelena; Polovic, Natalija

    2016-01-30

    The physiological role of fig latex is to protect the plant from pathogens. Latex is a rich source of proteases, predominantly ficin. Fig latex also contains collagenolytic protease and chitinolytic enzymes. Our aim was to investigate changes in protein composition, enzyme and antifungal activities of fig latex during fruit ripening. Comparison of latex samples in different time periods showed a uniform increase of protein concentration in chronological order. The content of collagenolytic protease did not differ significantly in the latex samples, while the content of ficin decreased. Ficin-specific activity towards casein was the highest at the beginning of fruit development (about 80 U mg(-1)). Specific milk clotting activity increased as well as the abundance of casein band in the clots. Specific chitinolytic activity at the beginning of flowering was 6.5 times higher than the activity in the period when fruits are ripe. Antifungal activity is the most extensive in spring. Ficin forms with different casein specificities are present in different proportions during fruit ripening, which is of importance for applications in the dairy industry. The protection mechanism against insects and fungi, which relies on chitinolytic activity, is the most important in the early phases of flowering and is replaced with other strategies over time. © 2015 Society of Chemical Industry.

  8. Serine Proteolytic Pathway Activation Reveals an Expanded Ensemble of Wound Response Genes in Drosophila

    PubMed Central

    Patterson, Rachel A.; Juarez, Michelle T.; Hermann, Anita; Sasik, Roman; Hardiman, Gary; McGinnis, William

    2013-01-01

    After injury to the animal epidermis, a variety of genes are transcriptionally activated in nearby cells to regenerate the missing cells and facilitate barrier repair. The range and types of diffusible wound signals that are produced by damaged epidermis and function to activate repair genes during epidermal regeneration remains a subject of very active study in many animals. In Drosophila embryos, we have discovered that serine protease function is locally activated around wound sites, and is also required for localized activation of epidermal repair genes. The serine protease trypsin is sufficient to induce a striking global epidermal wound response without inflicting cell death or compromising the integrity of the epithelial barrier. We developed a trypsin wounding treatment as an amplification tool to more fully understand the changes in the Drosophila transcriptome that occur after epidermal injury. By comparing our array results with similar results on mammalian skin wounding we can see which evolutionarily conserved pathways are activated after epidermal wounding in very diverse animals. Our innovative serine protease-mediated wounding protocol allowed us to identify 8 additional genes that are activated in epidermal cells in the immediate vicinity of puncture wounds, and the functions of many of these genes suggest novel genetic pathways that may control epidermal wound repair. Additionally, our data augments the evidence that clean puncture wounding can mount a powerful innate immune transcriptional response, with different innate immune genes being activated in an interesting variety of ways. These include puncture-induced activation only in epidermal cells in the immediate vicinity of wounds, or in all epidermal cells, or specifically in the fat body, or in multiple tissues. PMID:23637905

  9. Chronic Alcohol Intoxication Is Not Accompanied by an Increase in Calpain Proteolytic Activity in Cardiac Muscle of Rats.

    PubMed

    Gritsyna, Yu V; Salmov, N N; Bobylev, A G; Fadeeva, I S; Fesenko, N I; Sadikova, D G; Kukushkin, N I; Podlubnaya, Z A; Vikhlyantsev, I M

    2017-02-01

    Enzymatic activity of Ca2+-dependent calpain proteases as well as the content and gene expression of μ-calpain (activated by micromolar calcium ion concentrations), calpastatin (inhibitor of calpains), and titin (substrate for calpains) were investigated in cardiac muscles of rats subjected to chronic alcoholization for 3 and 6 months. There was no increase in the "heart weight/body weight" parameter indicating development of heart hypertrophy in the alcoholized rats, while a decreasing trend was observed for this parameter in the rats after 6-month modeling of alcoholic cardiomyopathy, which indicated development of atrophic changes in the myocardium. Fluorometric measurements conducted using the Calpain Activity Assay Kit did not reveal any changes in total calpain activity in protein extracts of cardiac muscles of the rats alcoholized for 3 and 6 months. Western blot analysis did not show reliable changes in the contents of μ-calpain and calpastatin, and SDS-PAGE did not reveal any decrease in the titin content in the myocardium of rats after the chronic alcohol intoxication. Autolysis of μ-calpain was also not verified, which could indicate that proteolytic activity of this enzyme in myocardium of chronically alcoholized rats is not enhanced. Using Pro-Q Diamond staining, changes in phosphorylation level of titin were not detected in cardiac muscle of rats after chronic alcoholization during three and six months. A decrease in µ-calpain and calpastatin mRNA content (~1.3-fold, p ≤ 0.01 and ~1.9-fold, p ≤ 0.01, respectively) in the myocardium of rats alcoholized for 3 months and decrease in calpastatin mRNA (~1.4-fold, p ≤ 0.01) in animals alcoholized for 6 months was demonstrated using real-time PCR. These results indicate negative effect of chronic alcohol intoxication on expression of the abovementioned genes.

  10. Searching for discrimination rules in protease proteolytic cleavage activity using genetic programming with a min-max scoring function.

    PubMed

    Yang, Zheng Rong; Thomson, Rebecca; Hodgman, T Charles; Dry, Jonathan; Doyle, Austin K; Narayanan, Ajit; Wu, XiKun

    2003-11-01

    This paper presents an algorithm which is able to extract discriminant rules from oligopeptides for protease proteolytic cleavage activity prediction. The algorithm is developed using genetic programming. Three important components in the algorithm are a min-max scoring function, the reverse Polish notation (RPN) and the use of minimum description length. The min-max scoring function is developed using amino acid similarity matrices for measuring the similarity between an oligopeptide and a rule, which is a complex algebraic equation of amino acids rather than a simple pattern sequence. The Fisher ratio is then calculated on the scoring values using the class label associated with the oligopeptides. The discriminant ability of each rule can therefore be evaluated. The use of RPN makes the evolutionary operations simpler and therefore reduces the computational cost. To prevent overfitting, the concept of minimum description length is used to penalize over-complicated rules. A fitness function is therefore composed of the Fisher ratio and the use of minimum description length for an efficient evolutionary process. In the application to four protease datasets (Trypsin, Factor Xa, Hepatitis C Virus and HIV protease cleavage site prediction), our algorithm is superior to C5, a conventional method for deriving decision trees.

  11. Identification of a Mutation Causing Deficient BMP1/mTLD Proteolytic Activity in Autosomal Recessive Osteogenesis Imperfecta

    PubMed Central

    Martínez-Glez, Víctor; Valencia, Maria; Caparrós-Martín, José A.; Aglan, Mona; Temtamy, Samia; Tenorio, Jair; Pulido, Veronica; Lindert, Uschi; Rohrbach, Marianne; Eyre, David; Giunta, Cecilia; Lapunzina, Pablo; Ruiz-Perez, Victor L.

    2013-01-01

    Herein, we have studied a consanguineous Egyptian family with two children diagnosed with severe autosomal recessive osteogenesis imperfecta (AR-OI) and a large umbilical hernia. Homozygosity mapping in this family showed lack of linkage to any of the previously known AR-OI genes, but revealed a 10.27 MB homozygous region on chromosome 8p in the two affected sibs, which comprised the procollagen I C-terminal propeptide (PICP) endopeptidase gene BMP1. Mutation analysis identified both patients with a Phe249Leu homozygous missense change within the BMP1 protease domain involving a residue, which is conserved in all members of the astacin group of metalloproteases. Type I procollagen analysis in supernatants from cultured fibroblasts demonstrated abnormal PICP processing in patient-derived cells consistent with the mutation causing decreased BMP1 function. This was further confirmed by overexpressing wild type and mutant BMP1 longer isoform (mammalian Tolloid protein [mTLD]) in NIH3T3 fibroblasts and human primary fibroblasts. While overproduction of normal mTLD resulted in a large proportion of proα1(I) in the culture media being C-terminally processed, proα1(I) cleavage was not enhanced by an excess of the mutant protein, proving that the Phe249Leu mutation leads to a BMP1/mTLD protein with deficient PICP proteolytic activity. We conclude that BMP1 is an additional gene mutated in AR-OI. PMID:22052668

  12. Cell homeostasis in a Leishmania major mutant overexpressing the spliced leader RNA is maintained by an increased proteolytic activity.

    PubMed

    Toledo, Juliano S; Ferreira, Tiago R; Defina, Tânia P A; Dossin, Fernando de M; Beattie, Kenneth A; Lamont, Douglas J; Cloutier, Serge; Papadopoulou, Barbara; Schenkman, Sergio; Cruz, Angela K

    2010-10-01

    Although several stage-specific genes have been identified in Leishmania, the molecular mechanisms governing developmental gene regulation in this organism are still not well understood. We have previously reported an attenuation of virulence in Leishmania major and L. braziliensis carrying extra-copies of the spliced leader RNA gene. Here, we surveyed the major differences in proteome and transcript expression profiles between the spliced leader RNA overexpressor and control lines using two-dimensional gel electrophoresis and differential display reverse transcription PCR, respectively. Thirty-nine genes related to stress response, cytoskeleton, proteolysis, cell cycle control and proliferation, energy generation, gene transcription, RNA processing and post-transcriptional regulation have abnormal patterns of expression in the spliced leader RNA overexpressor line. The evaluation of proteolytic pathways in the mutant revealed a selective increase of cysteine protease activity and an exacerbated ubiquitin-labeled protein population. Polysome profile analysis and measurement of cellular protein aggregates showed that protein translation in the spliced leader RNA overexpressor line is increased when compared to the control line. We found that L. major promastigotes maintain homeostasis in culture when challenged with a metabolic imbalance generated by spliced leader RNA surplus through modulation of intracellular proteolysis. However, this might interfere with a fine-tuned gene expression control necessary for the amastigote multiplication in the mammalian host.

  13. p97-dependent retrotranslocation and proteolytic processing govern formation of active Nrf1 upon proteasome inhibition.

    PubMed

    Radhakrishnan, Senthil K; den Besten, Willem; Deshaies, Raymond J

    2014-01-01

    Proteasome inhibition elicits an evolutionarily conserved response wherein proteasome subunit mRNAs are upregulated, resulting in recovery (i.e., 'bounce-back') of proteasome activity. We previously demonstrated that the transcription factor Nrf1/NFE2L1 mediates this homeostatic response in mammalian cells. We show here that Nrf1 is initially translocated into the lumen of the ER, but is rapidly and efficiently retrotranslocated to the cytosolic side of the membrane in a manner that depends on p97/VCP. Normally, retrotranslocated Nrf1 is degraded promptly by the proteasome and active species do not accumulate. However, in cells with compromised proteasomes, retrotranslocated Nrf1 escapes degradation and is cleaved N-terminal to Leu-104 to yield a fragment that is no longer tethered to the ER membrane. Importantly, this cleavage event is essential for Nrf1-dependent activation of proteasome gene expression upon proteasome inhibition. Our data uncover an unexpected role for p97 in activation of a transcription factor by relocalizing it from the ER lumen to the cytosol. DOI: http://dx.doi.org/10.7554/eLife.01856.001.

  14. Proteolytic activity alterations resulting from force-feeding in Muscovy and Pekin ducks.

    PubMed

    Awde, S; Marty-Gasset, N; Wilkesman, J; Rémignon, H

    2013-11-01

    We investigated liver protease activity in force-fed and non-force-fed ducks using zymography gels to better understand mechanisms underlying liver steatosis in palmipeds. Male Muscovy and Pekin ducks were slaughtered before and after a short period (13 d) while they were conventionally fed or force fed. The force-fed regimen contained a high level of carbohydrates and was delivered in large doses. Main hepatic proteases (matrix metalloprotease-2, calpains, and cathepsins) were extracted from raw liver and specifically activated within electrophoretic gels. Both force-fed Muscovy and Pekin ducks presented higher liver weights and BW associated with lower matrix metalloprotease-2 and m-calpain hepatic activities. On the other hand, hepatic cathepsin activity was not affected by force feeding. It was concluded that Muscovy and Pekin duck hepatic proteases are affected similarly by the force feeding. Thus, this cannot explain differences observed between Muscovy and Pekin ducks regarding their ability to develop hepatic steatosis generally reported in literature.

  15. Visualization of proteolytic activity associated with the apoptotic response in cancer cells

    NASA Astrophysics Data System (ADS)

    Tice, Brian George

    Caspases execute programmed cell death, where low levels of caspase activity are linked to cancer. Chemotherapies utilize induction of apoptosis as a key mechanism for cancer treatment, where caspase-3 is a major player involved in dismantling these aberrant cells. The ability to sensitively measure the initial caspase-3 cleavage events during apoptosis is important for understanding the initiation of this complex cellular process, however, current ensemble methods are not sensitive enough to measure single cleavage events in cells. By utilizing the optical properties of plasmon coupling, peptide-linked gold nanoparticles were developed to enable single molecule imaging of caspase-3 activity in two different cancer systems. Au crown nanoparticles were assembled in a multimeric fashion to overcome the high and heterogeneous background scattering of live cells. In a colon cancer (SW620) cell line challenged with tumor necrosis factor-alpha (TNF-alpha), single molecule trajectories show early stage caspase-3 activation within minutes, which was not detectable by ensemble assays until 23 hours. Variability in caspase-3 activation among the population of cells was identified and likely a result of each cell's specific resistance to death receptor-induced apoptosis. Following these studies, improvements by way of sensitivity and selectivity were tailored into an improved nanosensor construct. Au nanoshell dimers were prepared as a comparably bright construct with 1) reduced heterogeneity compared to the synthesis of the crown nanoparticles and 2) a peptide sequence highly selective for caspase-3. Chronic myeloid leukemia (CML) K562 cells were assessed for their early apoptotic response upon treatment with dasatinib, a clinically approved tyrosine kinase inhibitor that specifically targets BCR-ABL. It has been demonstrated that inhibition of BCR-ABL by dasatinib commits K562 cells to apoptosis. Single molecule experiments with Au nanoshell dimers show caspase-3 activation

  16. Extracellular proteolytic activity and molecular analysis of Microsporum canis strains isolated from symptomatic and asymptomatic cats.

    PubMed

    Viani, Flávio Cesar; Cazares Viani, Paula Regina; Gutierrez Rivera, Irma Nelly; Gonçalves da Silva, Eriques; Rodrigues Paula, Claudete; Gambale, Walderez

    2007-03-01

    Microsporum canis is the main zoophylic dermatophyte in dogs and cats, and it is also an important zoonotic agent. The literature showed that cats are asymptomatic carriers of M. canis. This is apparently due to host resistance and/or the presence of strains with lower virulence. This study was aimed to evaluate the keratinolytic, elastinolytic and collagenolytic activities of M. canis strains and their relationship with symptomatic and asymptomatic cats. In addition, these strains were analysed by RFLP. The strains isolated from cats with clinical dermatophytosis had higher keratinase and elastase activity than those isolated from asymptomatic animals (p minus than 0.05). There were not differences in RFLP patterns based on Hind III digestion.

  17. Proteolytic Degradation of SCOP in the Hippocampus Contributes to Activation of MAP Kinase and Memory

    PubMed Central

    Shimizu, Kimiko; Phan, Trongha; Mansuy, Isabelle; Storm, Daniel R.

    2007-01-01

    Summary Because activation of Erk1/2 MAP kinase (MAPK) is critical for hippocampus-dependent memory, there is considerable interest in mechanisms for regulation of MAPK during memory formation. Here we report that MAPK and CREB-mediated transcription are negatively regulated by SCOP (SCN Circadian Oscillatory Protein) and that SCOP is proteolyzed by calpain when hippocampal neurons are stimulated by BDNF, KCl depolarization, or NMDA. Moreover, training for novel object memory decreases SCOP in the hippocampus. To determine if hippocampus-dependent memory is influenced by SCOP in vivo, we generated a transgenic mouse strain for the inducible overexpression of SCOP in the forebrain. Overexpression of SCOP completely blocked memory for novel objects. We conclude that degradation of SCOP by calpain contributes to activation of MAPK during memory formation. PMID:17382888

  18. Proteolytic activity of Plasmodium falciparum subtilisin-like protease 3 on parasite profilin, a multifunctional protein.

    PubMed

    Alam, Asrar; Bhatnagar, Raj K; Relan, Udbhav; Mukherjee, Paushali; Chauhan, Virander S

    2013-10-01

    Subtilisin-like proteases of malaria parasite Plasmodium falciparum (PfSUB1, 2 and 3) are expressed at late asexual blood stages. PfSUB1 and 2 are considered important drug targets due to their essentiality for parasite blood stages and role in merozoite egress and invasion of erythrocytes. We have earlier shown the in vitro serine protease activity of PfSUB3 and its localization at asexual blood stages. In this study, we attempted to identify the biological substrate(s) of PfSUB3 and found parasite profilin (PfPRF) as a substrate of the protease. Eukaryotic profilins are multifunctional proteins with primary role in regulation of actin filament assembly. PfPRF possesses biochemical features of eukaryotic profilins and its rodent ortholog is essential in blood stages. Profilin from related apicomplexan parasite Toxoplasma gondii (TgPRF) is known to be involved in parasite motility, host cell invasion, active egress from host cell, immune evasion and virulence in mice. In this study, mature PfSUB3 proteolysed recombinant PfPRF in a dose-dependent manner in in vitro assays. Recombinant PfPRF was assessed for its proinflammatory activity and found to induce high level of TNF-α and low but significant level of IL-12 from mouse bone marrow-derived dendritic cells. Proteolysis of PfPRF by PfSUB3 is suggestive of the probable role of the protease in the processes of motility, virulence and immune evasion.

  19. Diverse impact of acute and long-term extracellular proteolytic activity on plasticity of neuronal excitability.

    PubMed

    Wójtowicz, Tomasz; Brzdąk, Patrycja; Mozrzymas, Jerzy W

    2015-01-01

    Learning and memory require alteration in number and strength of existing synaptic connections. Extracellular proteolysis within the synapses has been shown to play a pivotal role in synaptic plasticity by determining synapse structure, function, and number. Although synaptic plasticity of excitatory synapses is generally acknowledged to play a crucial role in formation of memory traces, some components of neural plasticity are reflected by nonsynaptic changes. Since information in neural networks is ultimately conveyed with action potentials, scaling of neuronal excitability could significantly enhance or dampen the outcome of dendritic integration, boost neuronal information storage capacity and ultimately learning. However, the underlying mechanism is poorly understood. With this regard, several lines of evidence and our most recent study support a view that activity of extracellular proteases might affect information processing in neuronal networks by affecting targets beyond synapses. Here, we review the most recent studies addressing the impact of extracellular proteolysis on plasticity of neuronal excitability and discuss how enzymatic activity may alter input-output/transfer function of neurons, supporting cognitive processes. Interestingly, extracellular proteolysis may alter intrinsic neuronal excitability and excitation/inhibition balance both rapidly (time of minutes to hours) and in long-term window. Moreover, it appears that by cleavage of extracellular matrix (ECM) constituents, proteases may modulate function of ion channels or alter inhibitory drive and hence facilitate active participation of dendrites and axon initial segments (AISs) in adjusting neuronal input/output function. Altogether, a picture emerges whereby both rapid and long-term extracellular proteolysis may influence some aspects of information processing in neurons, such as initiation of action potential, spike frequency adaptation, properties of action potential and dendritic

  20. Diverse impact of acute and long-term extracellular proteolytic activity on plasticity of neuronal excitability

    PubMed Central

    Wójtowicz, Tomasz; Brzdąk, Patrycja; Mozrzymas, Jerzy W.

    2015-01-01

    Learning and memory require alteration in number and strength of existing synaptic connections. Extracellular proteolysis within the synapses has been shown to play a pivotal role in synaptic plasticity by determining synapse structure, function, and number. Although synaptic plasticity of excitatory synapses is generally acknowledged to play a crucial role in formation of memory traces, some components of neural plasticity are reflected by nonsynaptic changes. Since information in neural networks is ultimately conveyed with action potentials, scaling of neuronal excitability could significantly enhance or dampen the outcome of dendritic integration, boost neuronal information storage capacity and ultimately learning. However, the underlying mechanism is poorly understood. With this regard, several lines of evidence and our most recent study support a view that activity of extracellular proteases might affect information processing in neuronal networks by affecting targets beyond synapses. Here, we review the most recent studies addressing the impact of extracellular proteolysis on plasticity of neuronal excitability and discuss how enzymatic activity may alter input-output/transfer function of neurons, supporting cognitive processes. Interestingly, extracellular proteolysis may alter intrinsic neuronal excitability and excitation/inhibition balance both rapidly (time of minutes to hours) and in long-term window. Moreover, it appears that by cleavage of extracellular matrix (ECM) constituents, proteases may modulate function of ion channels or alter inhibitory drive and hence facilitate active participation of dendrites and axon initial segments (AISs) in adjusting neuronal input/output function. Altogether, a picture emerges whereby both rapid and long-term extracellular proteolysis may influence some aspects of information processing in neurons, such as initiation of action potential, spike frequency adaptation, properties of action potential and dendritic

  1. Proteolytic Activity of Human Lymphoid Tumor Cells. Correlation with Tumor Progression

    PubMed Central

    Ribatti, Domenico; Ria, Roberto; Pellegrino, Antonio; Bruno, Michele; Merchionne, Francesca; Dammacco, Franco

    2000-01-01

    Matrix metalloproteinase (MMP) expression and production are associated with advanced-stage tumor and contribute to tumor progression, invasion and metastases. The current study was designed to determine the expression and production of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) by human lymphoid tumor cells. Changes in expression and production were also investigated during tumor progression of multiple myeloma and mycosis fungoides. In situ hybridization analysis revealed that lymphoblastic leukemia B cells (SB cell line), multiple myeloma (MM) cells (U266 cell line) and lymphoblastic leukemia T cells (CEM and Jurkat cell lines) express constitutively the mRNA for MMP-2 and/or MMP-9. We demonstrated by gelatin-zymography of cell culture medium that both enzymes were secreted in their cleaved (activated) form. In situ hybridization of bone marrow plasma cells and gelatin- zymography of the medium showed that patients with active MM (diagnosis, relapse, leukemic progression) express higher levels of MMP-2 mRNA and protein than patients with non-active MM (complete/objective response, plateau) and with monoclonal gammopathies of undetermined significance (MGUS). MMP-9 expression and secretion was similar in all patient groups. In patients with mycosis fungoides (MF), the expression of MMP-2 and MMP-9 mRNAs was significantly upregulated with advancing stage, in terms of lesions both positive for one of two mRNAs and with the greatest intensity of expression. Besides MF cells, the MMP-2 and/or MMP-9 mRNAs were expressed by some stromal cell populations (microvascular endothelial cells, fibroblasts, macrophages), suggesting that these cells cooperate in the process of tumor invasion. Our studies identify MMPs as an important class of proteinases involved in the extracellular matrix (ECM) degradation by human lymphoid tumors, and suggest that MMPs inhibitors may lead to important new treatment for their control. PMID:11097203

  2. Clinical Severity of β-thalassaemia/Hb E Disease Is Associated with Differential Activities of the Calpain-Calpastatin Proteolytic System

    PubMed Central

    Sukati, Suriyan; Svasti, Saovaros; Stifanese, Roberto; Averna, Monica; Panutdaporn, Nantika; Penglong, Tipparat; Melloni, Edon; Fucharoen, Suthat; Katzenmeier, Gerd

    2012-01-01

    Earlier observations in the literature suggest that proteolytic degradation of excess unmatched α-globin chains reduces their accumulation and precipitation in β-thalassaemia erythroid precursor cells and have linked this proteolytic degradation to the activity of calpain protease. The aim of this study was to correlate the activity of calpain and its inhibitor, calpastatin, with different degrees of disease severity in β-thalassaemia. CD34+ cells were enriched from peripheral blood of healthy individuals (control group) and patients with mild and severe clinical presentations of β0-thalassaemia/Hb E disease. By ex vivo cultivation promoting erythroid cell differentiation for 7 days, proerythroblasts, were employed for the functional characterization of the calpain-calpastatin proteolytic system. In comparison to the control group, enzymatic activity and protein amounts of μ-calpain were found to be more than 3-fold increased in proerythroblasts from patients with mild clinical symptoms, whereas no significant difference was observed in patients with severe clinical symptoms. Furthermore, a 1.6-fold decrease of calpastatin activity and 3.2-fold accumulation of a 34 kDa calpain-mediated degradation product of calpastatin were observed in patients with mild clinical symptoms. The increased activity of calpain may be involved in the removal of excess α-globin chains contributing to a lower degree of disease severity in patients with mild clinical symptoms. PMID:22615919

  3. Cell growth and proteolytic activity of Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus delbrueckii ssp. bulgaricus, and Streptococcus thermophilus in milk as affected by supplementation with peptide fractions.

    PubMed

    Gandhi, Akanksha; Shah, Nagendra P

    2014-12-01

    The present investigation examined the effects of supplementation of milk peptide fractions produced by enzymatic hydrolysis on the fermentation of reconstituted skim milk (RSM). Changes in pH, cell growth, proteolytic activity, and angiotensin-converting enzyme (ACE)-inhibitory activity were monitored during fermentation of RSM by pure cultures of Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus delbrueckii ssp. bulgaricus, and Streptococcus thermophilus. The study showed that supplementation with peptide fractions of different molecular weights did not significantly affect the bacterial growth in RSM. All bacteria showed an increased proteolytic activity in RSM supplemented with large peptides (>10 kDa), and L. helveticus in general exhibited the highest proteolytic activity among the bacteria studied. The ACE-inhibitory activity was observed to be the maximum in RSM supplemented with larger peptides (>10 kDa) for all bacteria. The results suggest that proteolysis by bacteria leads to increased production of ACE-inhibitory peptides compared to the supplemented peptides produced by enzymatic hydrolysis.

  4. Small Molecule Inhibitors of Bacillus anthracis Protective Antigen Proteolytic Activation and Oligomerization

    PubMed Central

    Wein, Alexander N.; Williams, Brian N.; Liu, Shihui; Ermolinsky, Boris; Provenzano, Daniele; Abagyan, Ruben; Orry, Andrew; Leppla, Stephen H.; Peredelchuk, Michael

    2012-01-01

    Protective antigen (PA), lethal factor, and edema factor, the protein toxins of Bacillus anthracis, are among its most important virulence factors and play a key role in infection. We performed a virtual ligand screen of a library of 10,000 members to identify compounds predicted to bind to PA and prevent its oligomerization. Four of these compounds slowed PA association in a FRET-based oligomerization assay, and two of those protected cells from intoxication at concentrations of 1–10 μM. Exploration of the protective mechanism by Western blot showed decreased SDS-resistant PA oligomer on cells, and surprisingly, decreased amounts of activated PA. In vitro assays showed that one of the inhibitors blocked furin-mediated cleavage of PA, apparently through its binding to the PA substrate. Thus, we have identified inhibitors that can independently block both PA’s cleavage by furin and its subsequent oligomerization. Lead optimization on these two backbones may yield compounds with high activity and specificity for the anthrax toxins. PMID:22954387

  5. Calcium chloride marination effects on beef steak tenderness and calpain proteolytic activity.

    PubMed

    Whipple, G; Koohmaraie, M

    1993-01-01

    A study was conducted in three phases to examine the effect of calcium chloride marination on tenderness. Steaks obtained 5 days postmortem were marinated in a 150 mM calcium chloride solution for 24 h and 48 h in phase 1, and for 48 h in phases 2 and 3. The steaks utilized were obtained from mature cows 8-11 years of age-phase 1; four control and four β-agonist fed steers-phase 2; and three Peidmontese and two Nelore 18-month-old steers-phase 3. Data were analyzed by analysis of variance for a split-plot design. In phase 1, marination failed to improve (P > 0·05) shear force values. However, shear force values were less than 5 kg which was uncommonly low for mature cows. In phase 2, marination improved (P < 0·05) meat tenderness regardless of diet. Yet, the steaks from the β-agonist fed steers remained less tender, even after marination, than the steaks from the control steers. In phase 3, shear force requirements were decreased (P < 0·01) with marination. Also, the activities of m-calpain and calpastatin decreased (P < 0·05) with calcium marination. It appeared that the improvement in tenderness was through the activation of m-calpain. Copyright © 1992. Published by Elsevier Ltd.

  6. Bilayer Membrane Modulation of Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) Structure and Proteolytic Activity

    PubMed Central

    Cerofolini, Linda; Amar, Sabrina; Lauer, Janelle L.; Martelli, Tommaso; Fragai, Marco; Luchinat, Claudio; Fields, Gregg B.

    2016-01-01

    Cell surface proteolysis is an integral yet poorly understood physiological process. The present study has examined how the pericellular collagenase membrane-type 1 matrix metalloproteinase (MT1-MMP) and membrane-mimicking environments interplay in substrate binding and processing. NMR derived structural models indicate that MT1-MMP transiently associates with bicelles and cells through distinct residues in blades III and IV of its hemopexin-like domain, while binding of collagen-like triple-helices occurs within blades I and II of this domain. Examination of simultaneous membrane interaction and triple-helix binding revealed a possible regulation of proteolysis due to steric effects of the membrane. At bicelle concentrations of 1%, enzymatic activity towards triple-helices was increased 1.5-fold. A single mutation in the putative membrane interaction region of MT1-MMP (Ser466Pro) resulted in lower enzyme activation by bicelles. An initial structural framework has thus been developed to define the role(s) of cell membranes in modulating proteolysis. PMID:27405411

  7. cDNA sequence and chromosomal localization of human enterokinase, the proteolytic activator of trypsinogen.

    PubMed

    Kitamoto, Y; Veile, R A; Donis-Keller, H; Sadler, J E

    1995-04-11

    Enterokinase is a serine protease of the duodenal brush border membrane that cleaves trypsinogen and produces active trypsin, thereby leading to the activation of many pancreatic digestive enzymes. Overlapping cDNA clones that encode the complete human enterokinase amino acid sequence were isolated from a human intestine cDNA library. Starting from the first ATG codon, the composite 3696 nt cDNA sequence contains an open reading frame of 3057 nt that encodes a 784 amino acid heavy chain followed by a 235 amino acid light chain; the two chains are linked by at least one disulfide bond. The heavy chain contains a potential N-terminal myristoylation site, a potential signal anchor sequence near the amino terminus, and six structural motifs that are found in otherwise unrelated proteins. These domains resemble motifs of the LDL receptor (two copies), complement component Clr (two copies), the metalloprotease meprin (one copy), and the macrophage scavenger receptor (one copy). The enterokinase light chain is homologous to the trypsin-like serine proteinases. These structural features are conserved among human, bovine, and porcine enterokinase. By Northern blotting, a 4.4 kb enterokinase mRNA was detected only in small intestine. The enterokinase gene was localized to human chromosome 21q21 by fluorescence in situ hybridization.

  8. Real-time Catheter Molecular Sensing of Inflammation in Proteolytically Active Atherosclerosis

    PubMed Central

    Jaffer, Farouc A; Vinegoni, Claudio; John, Michael C; Aikawa, Elena; Gold, Herman K.; Finn, Aloke V; Ntziachristos, Vasilis; Libby, Peter; Weissleder, Ralph

    2009-01-01

    Background To enable intravascular detection of inflammation in atherosclerosis, we developed a near-infrared fluorescence (NIRF) catheter-based strategy to sense cysteine protease activity during vascular catheterization. Methods and Results The NIRF catheter was designed based on a clinical coronary artery guidewire. In phantom studies of NIR fluorescent plaques, blood produced only a mild (<30%) attenuation of the fluorescence signal compared to saline, affirming the favorable optical properties of the NIR window. Catheter evaluation in vivo utilized atherosclerotic rabbits (n=11). Rabbits received an injection of a cysteine protease-activatable NIRF imaging agent (Prosense750, excitation/emission 750/770 nm) or saline. Catheter pullbacks through the blood-filled iliac artery detected NIRF signals 24 hours after injection of the probe. In the protease agent group, the in vivo peak plaque target-to-background ratio (TBR) was 558% greater than controls (mean±SEM, 6.8±1.9 vs. 1.3±0.3, p<0.05). Ex vivo fluorescence reflectance imaging corroborated these results (TBR 10.3±1.8 agent vs. 1.8±0.3 saline, p<0.01). In the protease group only, saline flush-modulated NIRF signal profiles further distinguished atheromata from normal segments in vivo (p<0.01). Good correlation between the in vivo and ex vivo plaque TBR was present (r=0.82, p<0.01). Histopathological analyses demonstrated strong NIRF signal in plaques only from the protease agent group. NIRF signals colocalized with immunoreactive macrophages and the cysteine protease cathepsin B. Conclusions An intravascular fluorescence catheter can detect cysteine protease activity in vessels the size of human coronary arteries in real-time using an activatable NIRF agent. This strategy could aid in detecting inflammation and high-risk plaques in small-sized arteries. PMID:18852366

  9. Asthmatic bronchial epithelium activated by the proteolytic allergen Der p 1 increases selective dendritic cell recruitment.

    PubMed

    Pichavant, Muriel; Charbonnier, Anne-Sophie; Taront, Solenne; Brichet, Anne; Wallaert, Benoît; Pestel, Joel; Tonnel, André-Bernard; Gosset, Philippe

    2005-04-01

    Airway dendritic cells (DCs) are crucial for allergen-induced sensitization and inflammation in allergic asthma. After allergen challenge, an increased number of DCs is observed in airway epithelium from patients with allergy. Because Der p 1, a cysteine protease allergen from Dermatophagoides pteronyssinus , induces chemokine production by bronchial epithelial cells (BECs), the purpose of this investigation was to evaluate the capacity of BEC exposed to Der p 1 to recruit DCs. Chemotactic activity of BEAS-2B, a bronchial epithelial cell line, and BECs from nonatopic controls and patients with allergic asthma was evaluated on the migration of precursors, immature and mature monocyte-derived DCs (MDDCs), and CD34 + -derived Langerhans cells (LCs). C-C chemokine ligand (CCL)-2, CCL5, and C-X-C chemokine ligand 10 production by BEAS-2B and BEC was increased after Der p 1 exposure, whereas the proenzyme proDer p 1 devoid of enzymatic activity had no effect. Der p 1 stimulation of BEAS-2B and BEC from both groups increased significantly the recruitment of MDDC precursors, depending on CCL2, CCL5, and C-X-C chemokine ligand 10 production. In a reconstituted polarized epithelium, apical application of Der p 1 enhanced MDDC precursor migration into the epithelial layer. Moreover, Der p 1 stimulation of BEC from patients with asthma but not from controls increased the migration of LC precursors, mainly dependent on CCL20 secretion. No migration of immature and mature DCs was observed. These data confirmed that BECs participate in the homeostasis of the DC network present within the bronchial epithelium through the secretion of chemokines. In allergic asthma, upregulation of CCL20 production induced LC recruitment, the role of which remains to be determined.

  10. Proteolytic processing and deubiquitinating activity of papain-like proteases of human coronavirus NL63.

    PubMed

    Chen, Zhongbin; Wang, Yanhua; Ratia, Kiira; Mesecar, Andrew D; Wilkinson, Keith D; Baker, Susan C

    2007-06-01

    Human coronavirus NL63 (HCoV-NL63), a common human respiratory pathogen, is associated with both upper and lower respiratory tract disease in children and adults. Currently, no antiviral drugs are available to treat CoV infections; thus, potential drug targets need to be identified and characterized. Here, we identify HCoV-NL63 replicase gene products and characterize two viral papain-like proteases (PLPs), PLP1 and PLP2, which process the viral replicase polyprotein. We generated polyclonal antisera directed against two of the predicted replicase nonstructural proteins (nsp3 and nsp4) and detected replicase proteins from HCoV-NL63-infected LLC-MK2 cells by immunofluorescence, immunoprecipitation, and Western blot assays. We found that HCoV-NL63 replicase products can be detected at 24 h postinfection and that these proteins accumulate in perinuclear sites, consistent with membrane-associated replication complexes. To determine which viral proteases are responsible for processing these products, we generated constructs representing the amino-terminal end of the HCoV-NL63 replicase gene and established protease cis-cleavage assays. We found that PLP1 processes cleavage site 1 to release nsp1, whereas PLP2 is responsible for processing both cleavage sites 2 and 3 to release nsp2 and nsp3. We expressed and purified PLP2 and used a peptide-based assay to identify the cleavage sites recognized by this enzyme. Furthermore, by using K48-linked hexa-ubiquitin substrate and ubiquitin-vinylsulfone inhibitor specific for deubiquitinating enzymes (DUBs), we confirmed that, like severe acute respiratory syndrome (SARS) CoV PLpro, HCoV-NL63 PLP2 has DUB activity. The identification of the replicase products and characterization of HCoV-NL63 PLP DUB activity will facilitate comparative studies of CoV proteases and aid in the development of novel antiviral reagents directed against human pathogens such as HCoV-NL63 and SARS-CoV.

  11. Proteolytic activity of Elapid and Viperid Snake venoms and its implication to digestion

    PubMed Central

    Bottrall, Joshua L; Madaras, Frank; Biven, Christopher D; Venning, Michael G; Mirtschin, Peter J

    2010-01-01

    Testing whether venoms may aid in digestion of the prey, eleven snake venoms were compared for the presence of proteases and endopeptidases that function in alkaline pH conditions. In vitro experiments examined the relative protease and endopeptidase activity of the venoms, which involved combining bovine muscle and snake venom in a buffered solution, encased within dialysis tubing. This mixture was then incubated at room temperature (∼20°C) for 24hr, with constant shaking. Bicinchoninic acid (BCA) assay and ninhydrin assay were used to determine peptide and amino acid concentrations. Histological and immunohistochemical investigations using N. kaouthia venom confirmed in vitro findings. Results show that B. arietans venom generated the highest amount of protein/peptides and amino acids in the dialysates, while O. scutellatus, N. ater niger and P. textilis venom did not show any significant protein degradation under alkaline conditions. Histological examination revealed varying degrees of muscle cell damage for each of the venom investigated, and the immunohistochemical study on N. kaouthia venom showed that the venom penetrated the muscle tissue to a significant degree. In vitro assays and histological results indicate that particular venoms may possess the ability to enhance digestion of bovine muscle tissue. PMID:21544178

  12. Proteolytic cleavage of ostrich and turkey pancreatic lipases: production of an active N-terminal domain.

    PubMed

    Ben Bacha, Abir; Fendri, Ahmed; Gargouri, Youssef; Mejdoub, Hafedh; Miled, Nabil

    2007-10-01

    The aim of this study was to check some biochemical and structural properties of ostrich and turkey pancreatic lipases (OPL and TPL, respectively). Limited proteolysis of OPL and TPL was performed in conditions similar to those reported for porcine pancreatic lipase. In the absence of bile salts and colipase, OPL failed to catalyze the hydrolysis of pure tributyrin or efficiently hydrolyze olive oil emulsion. When bile salts and colipase were preincubated with the substrate, the OPL kinetic behavior remained linear for more than 30 minutes. The enzyme presented a penetration power value into an egg phosphatidylcholine monomolecular film that was comparable to that of HPL and lower than that of TPL. Chymotrypsin, trypsin, and thermolysin were able to hydrolyze OPL and TPL in different ways. In both cases, only N-terminal fragments accumulated during the hydrolysis, whereas no C-terminal fragment was obtained in either case. Tryptic cleavage of OPL and TPL completely degraded the enzymes. Nevertheless, chymotryptic attack generated 35-kd and 43-kd forms for TPL and OPL, respectively. Interestingly, the OPL 43-kd form was inactive, whereas the TPL 35-kd protein conserved its lipolytic activity. OPL, TPL, and mammal pancreatic lipases share a high amino acid sequence homology. Further investigations are, however, needed to identify key residues involved in substrate recognition responsible for biochemical differences between the 2 classes of lipases.

  13. The proinflammatory cytokines interleukin-1α and tumor necrosis factor α promote the expression and secretion of proteolytically active cathepsin S from human chondrocytes.

    PubMed

    Caglič, Dejan; Repnik, Urška; Jedeszko, Christopher; Kosec, Gregor; Miniejew, Catherine; Kindermann, Maik; Vasiljeva, Olga; Turk, Vito; Wendt, K Ulrich; Sloane, Bonnie F; Goldring, Mary B; Turk, Boris

    2013-02-01

    Osteoarthritis and rheumatoid arthritis are destructive joint diseases that involve the loss of articular cartilage. Degradation of cartilage extracellular matrix is believed to occur due to imbalance between the catabolic and anabolic processes of resident chondrocytes. Previous work has suggested that various lysosomal cysteine cathepsins participate in cartilage degeneration; however, their exact roles in disease development and progression have not been elucidated. In order to study degradation processes under conditions resembling the in vivo milieu of the cartilage, we cultivated chondrocytes on a type II collagen-containing matrix. Stimulation of the cultivated chondrocytes with interleukin-1α and/or tumor necrosis factor α resulted in a time-dependent increase in cathepsin S expression and induced its secretion into the conditioned media. Using a novel bioluminescent activity-based probe, we were able to demonstrate a significant increase in proteolytic activity of cathepsin S in the conditioned media of proinflammatory cytokine-stimulated chondrocytes. For the first time, cathepsin S was demonstrated to be secreted from chondrocytes upon stimulation with the proinflammatory cytokines, and displayed proteolytic activity in culture supernatants. Its stability at neutral pH and potent proteolytic activity on extracellular matrix components mean that cathepsin S may contribute significantly to cartilage degradation and may thus be considered a potential drug target in joint diseases.

  14. Insulin infusion increases levels of free IGF-I and IGFBP-3 proteolytic activity in patients after surgery.

    PubMed

    Nygren, J; Carlsson-Skwirut, C; Brismar, K; Thorell, A; Ljungqvist, O; Bang, P

    2001-10-01

    We have studied the effects of insulin on the bioavailability of insulin-like growth factor (IGF) I in insulin-resistant patients after surgery. Serum levels of total IGF-I (tIGF-I), free IGF (fIGF)-I, fIGF-II, and IGF-binding protein (IGFBP) 1 and IGFBP-3 proteolytic activity (IGFBP-3-PA), determined on the day before surgery and on the 1st postoperative day, were related to insulin sensitivity measured by a hyperinsulinemic, normoglycemic clamp. Before surgery, the decreased tIGF-I (P < 0.05) in response to insulin infusion was accompanied by an 18% reduction of IGFBP-1 (P < 0.001), while IGFBP-3-PA remained unchanged. Levels of fIGF-I and fIGF-II were not changed by insulin infusions. After surgery, IGFBP-3-PA increased (P < 0.05) during insulin infusion, and this was associated with an increase in tIGF-I (P < 0.001) and fIGF-I (P < 0.01), while no significant change was found in fIGF-II. The reduction in IGFBP-1 in response to insulin infusion was not affected by surgery. The change in IGFBP-3-PA during insulin infusion after surgery was related to the corresponding change in fIGF-I (r(2) = 0.26, P < 0.05) and postoperative insulin sensitivity (r(2) = -0.22, P < 0.05). These data suggest that increased IGFBP-3-PA during insulin infusion after surgery governs the increased levels of fIGF-I, while insulin-induced suppression of IGFBP-1 was not affected by surgery. We propose that, in catabolic, postoperative patients, increased levels of insulin from exogenous or, possibly, endogenous sources (nutritionally induced) may be a signal to increase IGF-I bioavailability by increased expression of IGFBP-3-PA to counteract further deterioration in glucose metabolism.

  15. Elastinolytic and proteolytic enzymes.

    PubMed

    Kessler, Efrat; Safrin, Mary

    2014-01-01

    Pseudomonas aeruginosa secretes into its environment at least seven extracellular proteases: pseudolysin (LasB protease; elastase), aeruginolysin (alkaline proteinase), staphylolysin (staphylolytic endopeptidase; LasA protease), lysyl endopeptidase (protease IV; PrpL), PASP (P. aeruginosa small protease), LepA (Large ExoProtease A), and an aminopeptidase. Their action on host proteins, both individually and synergistically, plays important roles in pathogenesis of P. aeruginosa infections. Methods to measure/detect their activities are fundamental for understanding their physiological functions, roles in pathogenesis, mechanisms of action, regulation, and secretion. Most assays for determination/detection of proteolytic activity employ modified/non-modified casein or gelatin as substrates. In the quantitative assay, fragments generated from azocasein are separated from undigested substrate by trichloroacetic acid precipitation and their absorbance is measured. In non-quantitative assays, proteolytic activity is detected as clearing zones around bacterial growth or samples of culture supernatants on casein containing solid media formed due to local casein degradation. In zymography, individual proteases are detected as clear bands in gelatin/casein containing gels after SDS-PAGE separation, renaturation and protein staining. The elastinolytic capacity of P. aeruginosa is reflected by clearing zones on nutrient agar plates containing insoluble elastin instead of casein. Mueller-Hinton agar plates on which S. aureus cells are grown as a lawn are used to assess the susceptibility of S. aureus isolates to staphylolysin. A clear zone around a staphylolysin-containing sample indicates inhibition of S. aureus growth. Methods for measuring the activity of individual proteases are based on their cleavage specificity. These include assays of elastinolytic activity of pseudolysin and/or staphylolysin using elastin-Congo red as a substrate, a method for determination of

  16. Proteolytic cleavage of chemerin protein is necessary for activation to the active form, Chem157S, which functions as a signaling molecule in glioblastoma.

    PubMed

    Yamaguchi, Yasuto; Du, Xiao-Yan; Zhao, Lei; Morser, John; Leung, Lawrence L K

    2011-11-11

    Chemerin is a chemoattractant involved in innate and adaptive immunity as well as an adipokine implicated in adipocyte differentiation. Chemerin circulates as an inactive precursor in blood whose bioactivity is closely regulated through proteolytic processing at its C terminus. We developed methodology for production of different recombinant chemerin isoforms (chem163S, chem157S, and chem155A) which allowed us to obtain large quantities of these proteins with purity of >95%. Chem158K was generated from chem163S by plasmin cleavage. Characterization by mass spectrometry and Edman degradation demonstrated that both the N and C termini were correct for each isoform. Ca(2+) mobilization assays showed that the EC(50) values for chem163S and chem158K were 54.2 ± 19.9 nm and 65.2 ± 13.2 nm, respectively, whereas chem157S had a ∼50-fold higher potency with an EC(50) of 1.2 ± 0.7 nm. Chem155A had no agonist activity and weak antagonist activity, causing a 50% reduction of chem157S activity at a molar ratio of 100:1. Similar results were obtained in a chemotaxis assay. Because chem158K is the dominant form in cerebrospinal fluid from patients with glioblastoma (GBM), we examined the significance of chemerin in GBM biology. In silico analysis showed chemerin mRNA was significantly increased in tissue from grade III and IV gliomas. Furthermore, U-87 MG cells, a human GBM line, express the chemerin receptors, chemokine-like receptor 1 and chemokine receptor-like 2, and chem157S triggered Ca(2+) flux. This study emphasized the necessity of appropriate C-terminal proteolytic processing to generate the likely physiologic form of active chemerin, chem157S, and suggested a possible role in malignant GBM.

  17. Production of barley endoprotease B2 in Pichia pastoris and its proteolytic activity against native and recombinant hordeins.

    PubMed

    Rosenkilde, Anne Lind; Dionisio, Giuseppe; Holm, Preben B; Brinch-Pedersen, Henrik

    2014-01-01

    Barley (Hordeum vulgare L.) cysteine proteases are of fundamental biological importance during germination but may also have a large potential as commercial enzyme. Barley cysteine endoprotease B2 (HvEPB2) was expressed in Pichia pastoris from a pPICZαA based construct encoding a HvEPB2 C-terminal truncated version (HvEPB2ΔC) and a proteolytic resistant His6 tag. Maximum yield was obtained after 4 days of induction. Recombinant HvEPB2ΔC (r-HvEPB2ΔC) was purified using a single step of Ni(2+)-affinity chromatography. Purified protein was evaluated by SDS-PAGE, Western blotting and activity assays. A purification yield of 4.26 mg r-HvEPB2ΔC per L supernatant was obtained. r-HvEPB2ΔC follows first order kinetics (Km=12.37 μM) for the substrate Z-Phe-Arg-pNA and the activity was significantly inhibited by the cysteine protease specific inhibitors E64 and leupeptin. The temperature optimum for r-HvEPB2ΔC was 60°C, thermal stability T50 value was 44°C and the pH optimum was 4.5. r-HvEPB2ΔC was incubated with native purified barley seed storage proteins for up to 48 h. After 12h, r-HvEPB2ΔC efficiently reduced the C and D hordeins almost completely, as evaluated by SDS-PAGE. The intensities of the B and γ hordein bands decreased continuously over the 48 h. No degradation occurred in the presence of E64. Recombinant hordeins (B1, B3 and γ1) were expressed in Escherichia coli. After 2h of incubation with r-HvEPB2ΔC, an almost complete degradation of γ1 and partial digests of hordein B1 and B3 were observed.

  18. Distinctive proteolytic activity of cell envelope proteinase of Lactobacillus helveticus isolated from airag, a traditional Mongolian fermented mare's milk.

    PubMed

    Miyamoto, Mari; Ueno, Hiroshi M; Watanabe, Masayuki; Tatsuma, Yumi; Seto, Yasuyuki; Miyamoto, Taku; Nakajima, Hadjime

    2015-03-16

    Airag is a traditional fermented milk of Mongolia that is usually made from raw mare's milk. Lactobacillus helveticus is one of the lactic acid bacteria most frequently isolated from airag. In this study, we investigated the genetic and physiological characteristics of L. helveticus strains isolated from airag and clarified their significance in airag by comparing them with strains from different sources. Six strains of L. helveticus were isolated from five home-made airag samples collected from different regions of Mongolia. The optimal temperature for acidification in skim milk was 30 to 35°C for all the Mongolian strains, which is lower than those for the reference strains (JCM 1554 and JCM 1120(T)) isolated from European cheeses. All of the strains had a prtH1-like gene encoding a variant type of cell envelope proteinase (CEP). The CEP amino acid sequence in Snow Brand Typeculture (SBT) 11087 isolated from airag shared 71% identity with PrtH of L. helveticus CNRZ32 (AAD50643.1) but 98% identity with PrtH of Lactobacillus kefiranofaciens ZW3 (AEG40278.1) isolated from a traditional fermented milk in Tibet. The proteolytic activities of the CEP from SBT11087 on artificial substrate (N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide) and pure casein were measured using an intact-cell degradation assay. The activity of the CEP from SBT11087 was observed to be weak and exhibited a lower optimal temperature (40°C) than those from the reference strains (45-50°C). The specificity of the SBT11087 CEP for αS1-casein was typical of the CEPs previously reported in L. helveticus, as determined through the degradation profiles obtained through gel electrophoresis and mass spectrometry analyses. In contrast, the degradation profile of β-casein revealed that the CEP of SBT11087 primarily hydrolyzes its C-terminal domain and hydrolyzed nine of the 16 cleavage sites shared among the CEPs of other L. helveticus strains. Thus, the CEP of SBT11087 is distinct from those from

  19. Proteolytic Cleavage Driven by Glycosylation*

    PubMed Central

    Kötzler, Miriam P.; Withers, Stephen G.

    2016-01-01

    Proteolytic processing of human host cell factor 1 (HCF-1) to its mature form was recently shown, unexpectedly, to occur in a UDP-GlcNAc-dependent fashion within the transferase active site of O-GlcNAc-transferase (OGT) (Lazarus, M. B., Jiang, J., Kapuria, V., Bhuiyan, T., Janetzko, J., Zandberg, W. F., Vocadlo, D. J., Herr, W., and Walker, S. (2013) Science 342, 1235–1239). An interesting mechanism involving formation and then intramolecular rearrangement of a covalent glycosyl ester adduct of the HCF-1 polypeptide was proposed to account for this unprecedented proteolytic activity. However, the key intermediate remained hypothetical. Here, using a model enzyme system for which the formation of a glycosyl ester within the enzyme active site has been shown unequivocally, we show that ester formation can indeed lead to proteolysis of the adjacent peptide bond, thereby providing substantive support for the mechanism of HCF-1 processing proposed. PMID:26515062

  20. Effect of retinal laser photocoagulation on the activity of metalloproteinases and the alpha(2)-macroglobulin proteolytic state in the vitreous of eyes with proliferative diabetic retinopathy.

    PubMed

    Sánchez, María C; Luna, Jose D; Barcelona, Pablo F; Gramajo, Ana L; Juarez, Patricio C; Riera, Clelia M; Chiabrando, Gustavo A

    2007-11-01

    Panretinal photocoagulation (PRP) reduces the incidence of severe visual loss in proliferative diabetic retinopathy (PDR). The aim of the study was to determine the effect of PRP on the activity of matrix metalloproteinase-2 (MMP-2) and MMP-9, and also on the alpha(2)-Macroglobulin (alpha(2)M) proteolytic state in the vitreous of eyes with PDR. Vitreous samples were obtained from patients undergoing vitrectomy for the treatment of retinal diseases: 17 with PDR and eight with idiopathic macular hole (MH). Qualitative evaluation of the MMP-2 and MMP-9 activation status was performed by gelatin zymography and quantitative assay was carried out for vitreous total protein content and alpha(2)M. The proteolytic state of alpha(2)M was evaluated by Western blotting. Although all vitreous samples contained proMMP-2, increased proMMP-9 and active MMP-9 were detected in PDR samples without PRP. In addition, after PRP the proMMP-9 activity was significantly decreased, whereas the proMMP-2 activity was not affected. Enhanced total protein and alpha(2)M concentrations were observed in all vitreous samples from PDR patients with and without previous PRP compared with samples from patients with MH. However, a differential proteolytic state of alpha(2)M, expressed as 180/85-90kDa ratio, was detected among PDR patients with and without PRP treatment. Whereas a low 180/85-90kDa ratio of alpha(2)M in vitreous of PDR patients without PRP was observed, a high proportion of 180kDa subunit was principally detected in PDR with PRP. These results demonstrate that PDR occurs with an enhanced activity of MMP-9 and activation of alpha(2)M by proteinases, which is reversed after PRP. In addition, we suggest that alpha(2)M plays a key role in the control and regulation of the ocular neovascularization involved in the pathogenesis of ischemic retinal diseases such as PDR.

  1. Demonstration of Proteolytic Activation of the Epithelial Sodium Channel (ENaC) by Combining Current Measurements with Detection of Cleavage Fragments

    PubMed Central

    Krappitz, Matteus; Korbmacher, Christoph; Haerteis, Silke

    2014-01-01

    The described methods can be used to investigate the effect of proteases on ion channels, receptors, and other plasma membrane proteins heterologously expressed in Xenopus laevis oocytes. In combination with site-directed mutagenesis, this approach provides a powerful tool to identify functionally relevant cleavage sites. Proteolytic activation is a characteristic feature of the amiloride-sensitive epithelial sodium channel (ENaC). The final activating step involves cleavage of the channel’s γ-subunit in a critical region potentially targeted by several proteases including chymotrypsin and plasmin. To determine the stimulatory effect of these serine proteases on ENaC, the amiloride-sensitive whole-cell current (ΔIami) was measured twice in the same oocyte before and after exposure to the protease using the two-electrode voltage-clamp technique. In parallel to the electrophysiological experiments, a biotinylation approach was used to monitor the appearance of γENaC cleavage fragments at the cell surface. Using the methods described, it was demonstrated that the time course of proteolytic activation of ENaC-mediated whole-cell currents correlates with the appearance of a γENaC cleavage product at the cell surface. These results suggest a causal link between channel cleavage and channel activation. Moreover, they confirm the concept that a cleavage event in γENaC is required as a final step in proteolytic channel activation. The methods described here may well be applicable to address similar questions for other types of ion channels or membrane proteins. PMID:25045853

  2. An upstream initiator caspase 10 of snakehead murrel Channa striatus, containing DED, p20 and p10 subunits: molecular cloning, gene expression and proteolytic activity.

    PubMed

    Arockiaraj, Jesu; Gnanam, Annie J; Muthukrishnan, Dhanaraj; Pasupuleti, Mukesh; Milton, James; Singh, Arun

    2013-02-01

    Caspase 10 (CsCasp10) was identified from a constructed cDNA library of freshwater murrel (otherwise called snakehead) Channa striatus. The CsCasp10 is 1838 base pairs (bp) in length and it is encoding 549 amino acid (aa) residues. CsCasp10 amino acid contains two death effector domains (DED) in the N-terminal at 2-77 and 87-154 and it contains caspase family p20 domain (large subunit) and caspase family p10 domain (small subunit) in the C-terminal at 299-425 and 449-536 respectively. Pairwise analysis of CsCasp10 showed the highest sequence similarity (79%) with caspase 10 of Paralichthys olivaceus. Moreover, the phylogenetic analysis showed that CsCasp10 is clustered together with other fish caspase 10, formed a sister group with caspase 10 from other lower vertebrates including amphibian, reptile and birds and finally clustered together with higher vertebrates such as mammals. Significantly (P < 0.05) highest CsCasp10 gene expression was noticed in gills and lowest in intestine. Furthermore, the CsCasp10 gene expression in C. striatus was up-regulated in gills by fungus Aphanomyces invadans and bacteria Aeromonas hydrophila induction. The proteolytic activity was analyzed using the purified recombinant CsCasp10 protein. The results showed the proteolytic activity of CsCasp10 for caspase 10 substrate was 2.5 units per μg protein. Moreover, the proteolytic activities of CsCasp10 in kidney and spleen induced by A. invadans and A. hydrophila stimulation were analyzed by caspase 10 activity assay kit. All these results showed that CsCasp10 are participated in immunity of C. striatus against A. invadans and A. hydrophila infection.

  3. Prediction of extracellular proteases of the human pathogen Helicobacter pylori reveals proteolytic activity of the Hp1018/19 protein HtrA.

    PubMed

    Löwer, Martin; Weydig, Christiane; Metzler, Dirk; Reuter, Andreas; Starzinski-Powitz, Anna; Wessler, Silja; Schneider, Gisbert

    2008-01-01

    Exported proteases of Helicobacter pylori (H. pylori) are potentially involved in pathogen-associated disorders leading to gastric inflammation and neoplasia. By comprehensive sequence screening of the H. pylori proteome for predicted secreted proteases, we retrieved several candidate genes. We detected caseinolytic activities of several such proteases, which are released independently from the H. pylori type IV secretion system encoded by the cag pathogenicity island (cagPAI). Among these, we found the predicted serine protease HtrA (Hp1019), which was previously identified in the bacterial secretome of H. pylori. Importantly, we further found that the H. pylori genes hp1018 and hp1019 represent a single gene likely coding for an exported protein. Here, we directly verified proteolytic activity of HtrA in vitro and identified the HtrA protease in zymograms by mass spectrometry. Overexpressed and purified HtrA exhibited pronounced proteolytic activity, which is inactivated after mutation of Ser205 to alanine in the predicted active center of HtrA. These data demonstrate that H. pylori secretes HtrA as an active protease, which might represent a novel candidate target for therapeutic intervention strategies.

  4. Prediction of Extracellular Proteases of the Human Pathogen Helicobacter pylori Reveals Proteolytic Activity of the Hp1018/19 Protein HtrA

    PubMed Central

    Löwer, Martin; Weydig, Christiane; Metzler, Dirk; Reuter, Andreas; Starzinski-Powitz, Anna

    2008-01-01

    Exported proteases of Helicobacter pylori (H. pylori) are potentially involved in pathogen-associated disorders leading to gastric inflammation and neoplasia. By comprehensive sequence screening of the H. pylori proteome for predicted secreted proteases, we retrieved several candidate genes. We detected caseinolytic activities of several such proteases, which are released independently from the H. pylori type IV secretion system encoded by the cag pathogenicity island (cagPAI). Among these, we found the predicted serine protease HtrA (Hp1019), which was previously identified in the bacterial secretome of H. pylori. Importantly, we further found that the H. pylori genes hp1018 and hp1019 represent a single gene likely coding for an exported protein. Here, we directly verified proteolytic activity of HtrA in vitro and identified the HtrA protease in zymograms by mass spectrometry. Overexpressed and purified HtrA exhibited pronounced proteolytic activity, which is inactivated after mutation of Ser205 to alanine in the predicted active center of HtrA. These data demonstrate that H. pylori secretes HtrA as an active protease, which might represent a novel candidate target for therapeutic intervention strategies. PMID:18946507

  5. Fermented Infant Formula Increases Ileal Protein Digestibility and Reduces Ileal Proteolytic Activity Compared with Standard and Hydrolyzed Infant Formulas in Piglets.

    PubMed

    Abrahamse, Evan; Huybers, Sylvie; Alles, Martine S; Renes, Ingrid B; Knol, Jan; Bouritius, Hetty; Ludwig, Thomas

    2015-07-01

    An infant formula that contained milk fermented by the bacteria Bifidobacterium breve and Streptococcus thermophilus (Lactofidus) was reported to alleviate functional digestive symptoms in infants. It was hypothesized that improved protein digestibility of the fermented infant formula could contribute to this effect. The aim of this study was to evaluate the protein digestibility of a specific fermented (FF), a standard (SF), and an extensively hydrolyzed protein (HF) formula. Four-week-old piglets (n = 7) were fitted with a T-cannula at the terminal ileum and received each formula in a Latin square design. FF, SF, and HF contained 11.7%, 9.3%, and 11.9% (w/w) crude protein; 1.5%, 5.4%, and 5.6% (w/w) fiber; and had a casein/whey ratio of 60:40, 50:50, and 0:100 per kilogram of powder, respectively. Ileal digesta were collected and analyzed for amino acids and proteolytic activity. FF had a significantly higher apparent ileal crude protein digestibility (92.1% ± 1.0%) than SF and HF (84.4% ± 1.0% and 83.9% ± 0.9%, respectively). FF also had a significantly higher dry matter digestibility than SF and HF. The ileal crude protein flow of FF was significantly lower than that of SF and HF. The ileal flow of FF total proteolytic activity was significantly lower than that of SF but not significantly different from that of HF (412 ± 163 kU/8 h vs. 1530 ± 163 and 703 ± 156 kU/8 h, respectively). The FF in piglets had a significantly higher apparent ileal crude protein digestibility than the SF and HF and displayed lower ileal proteolytic activity than the SF. Both effects may contribute to the alleviation of functional gastrointestinal symptoms reported in infants fed fermented infant milk formula. © 2015 American Society for Nutrition.

  6. Proteolytic properties of Funastrum clausum latex.

    PubMed

    Morcelle, Susana R; Caffini, Néstor O; Priolo, Nora

    2004-07-01

    As part of a screening of latex endopeptidases from plants growing in Argentina, the presence of proteolytic activity in the latex of Funastrum clausum stems is reported. The proteases present in the crude extract showed the main characteristics of the cysteine proteolytic class, i.e. optimum pH at alkaline range, isoelectric point (pI) higher than 9.0, and inhibition of proteolytic activity by thiol blocking reagents. A remarkable thermal stability was also evident in the crude extract. Endosterolytic preference tried on p-nitrophenyl esters of N-alpha-carbobenzoxy-L-amino acids was higher for the alanine, asparagine and tyrosine derivatives. Preliminary peptidase purification by two-step ionic exchange showed the presence of two proteolytic fractions with molecular masses of approximately 24.0 kDa according to SDS-PAGE.

  7. Campylobacter jejuni outer membrane vesicle-associated proteolytic activity promotes bacterial invasion by mediating cleavage of intestinal epithelial cell E-cadherin and occludin.

    PubMed

    Elmi, Abdi; Nasher, Fauzy; Jagatia, Heena; Gundogdu, Ozan; Bajaj-Elliott, Mona; Wren, Brendan; Dorrell, Nick

    2016-04-01

    Outer membrane vesicles (OMVs) play an important role in the pathogenicity of Gram-negative bacteria. Campylobacter jejuni produces OMVs that trigger IL-8, IL-6, hBD-3 and TNF-α responses from T84 intestinal epithelial cells and are cytotoxic to Caco-2 IECs and Galleria mellonella larvae. Proteomic analysis of 11168H OMVs identified the presence of three proteases, HtrA, Cj0511 and Cj1365c. In this study, 11168H OMVs were shown to possess proteolytic activity that was reduced by pretreatment with specific serine protease inhibitors. OMVs isolated from 11168H htrA, Cj0511 or Cj1365c mutants possess significantly reduced proteolytic activity. 11168H OMVs are able to cleave both E-cadherin and occludin, but this cleavage is reduced with OMVs pretreated with serine protease inhibitors and also with OMVs isolated from htrA or Cj1365c mutants. Co-incubation of T84 monolayers with 11168H OMVs results in a visible reduction in both E-cadherin and occludin. The addition of 11168H OMVs to the co-culture of live 11168H bacteria with T84 cells results in enhanced levels of bacterial adhesion and invasion in a time-dependent and dose-dependent manner. Further investigation of the cleavage of host cell structural proteins by C. jejuni OMVs should enhance our understanding of the interactions of this important pathogen with intestinal epithelial cells. © 2015 John Wiley & Sons Ltd.

  8. Proteolytic activation of pro-spätzle is required for the induced transcription of antimicrobial peptide genes in lepidopteran insects

    PubMed Central

    Wang, Yang; Cheng, Tingcai; Rayaprolu, Subrahmanyam; Zou, Zhen; Xia, Qingyou; Xiang, Zhonghuai; Jiang, Haobo

    2007-01-01

    Microbial infection leads to proteolytic activation of Drosophila spätzle, which binds to the toll receptor and induces the synthesis of immune proteins. To test whether or not this mechanism exists in lepidopteran insects, we cloned the cDNA of Bombyx mori spätzle-1 and overexpressed the full-length and truncated BmSpz1 cDNA in Escherichia coli. The insoluble fusion proteins were affinity-purified under denaturing condition. After the silkworm larvae were injected with renatured BmSpz1, mRNA levels of antimicrobial peptide genes greatly increased. Similar transcriptional up-regulation was also found in Manduca sexta. Injection of pro-BmSpz1 had no such effect. When pro-BmSpz1 and Micrococcus luteus were incubated with the plasma from M. sexta larvae, we detected proteolytic processing of pro-BmSpz1. These results suggest that active spätzle is required for the induced production of antimicrobial peptides in B. mori and M. sexta. PMID:17337053

  9. Kinetic characterization of trans-proteolytic activity of Chikungunya virus capsid protease and development of a FRET-based HTS assay

    PubMed Central

    Aggarwal, Megha; Sharma, Rajesh; Kumar, Pravindra; Parida, Manmohan; Tomar, Shailly

    2015-01-01

    Chikungunya virus (CHIKV) capsid protein (CVCP) is a serine protease that possesses cis-proteolytic activity essential for the structural polyprotein processing and plays a key role in the virus life cycle. CHIKV being an emerging arthropod-borne pathogenic virus, is a public health concern worldwide. No vaccines or specific antiviral treatment is currently available for chikungunya disease. Thus, it is important to develop inhibitors against CHIKV enzymes to block key steps in viral reproduction. In view of this, CVCP was produced recombinantly and purified to homogeneity. A fluorescence resonance energy transfer (FRET)-based proteolytic assay was developed for high throughput screening (HTS). A FRET peptide substrate (DABCYL-GAEEWSLAIE-EDANS) derived from the cleavage site present in the structural polyprotein of CVCP was used. The assay with a Z’ factor of 0.64 and coefficient of variation (CV) is 8.68% can be adapted to high throughput format for automated screening of chemical libraries to identify CVCP specific protease inhibitors. Kinetic parameters Km and kcat/Km estimated using FRET assay were 1.26 ± 0.34 μM and 1.11 × 103 M−1 sec−1 respectively. The availability of active recombinant CVCP and cost effective fluorogenic peptide based in vitro FRET assay may serve as the basis for therapeutics development against CHIKV. PMID:26439734

  10. A Prodomain Fragment from the Proteolytic Activation of Growth Differentiation Factor 11 Remains Associated with the Mature Growth Factor and Keeps It Soluble.

    PubMed

    Pepinsky, Blake; Gong, Bang-Jian; Gao, Yan; Lehmann, Andreas; Ferrant, Janine; Amatucci, Joseph; Sun, Yaping; Bush, Martin; Walz, Thomas; Pederson, Nels; Cameron, Thomas; Wen, Dingyi

    2017-08-22

    Growth differentiation factor 11 (GDF11), a member of the transforming growth factor β (TGF-β) family, plays diverse roles in mammalian development. It is synthesized as a large, inactive precursor protein containing a prodomain, pro-GDF11, and exists as a homodimer. Activation requires two proteolytic processing steps that release the prodomains and transform latent pro-GDF11 into active mature GDF11. In studying proteolytic activation in vitro, we discovered that a 6-kDa prodomain peptide containing residues 60-114, PDP60-114, remained associated with the mature growth factor. Whereas the full-length prodomain of GDF11 is a functional antagonist, PDP60-114 had no impact on activity. The specific activity of the GDF11/PDP60-114 complex (EC50 = 1 nM) in a SMAD2/3 reporter assay was identical to that of mature GDF11 alone. PDP60-114 improved the solubility of mature GDF11 at neutral pH. As the growth factor normally aggregates/precipitates at neutral pH, PDP60-114 can be used as a solubility-enhancing formulation. Expression of two engineered constructs with PDP60-114 genetically fused to the mature domain of GDF11 through a 2x or 3x G4S linker produced soluble monomeric products that could be dimerized through redox reactions. The construct with a 3x G4S linker retained 10% activity (EC50 = 10 nM), whereas the construct connected with a 2x G4S linker could only be activated (EC50 = 2 nM) by protease treatment. Complex formation with PDP60-114 represents a new strategy for stabilizing GDF11 in an active state that may translate to other members of the TGF-β family that form latent pro/mature domain complexes.

  11. Substitution of cysteine 192 in a highly conserved Streptococcus pyogenes extracellular cysteine protease (interleukin 1beta convertase) alters proteolytic activity and ablates zymogen processing.

    PubMed Central

    Musser, J M; Stockbauer, K; Kapur, V; Rudgers, G W

    1996-01-01

    Virtually all strains of the human pathogenic bacterium Streptococcus pyogenes express a highly conserved extracellular cysteine protease. The protein is made as an inactive zymogen of 40,000 Da and undergoes autocatalytic truncation to result in a 28,000-Da active protease. Numerous independent lines of investigation suggest that this enzyme participates in one or more phases of host-parasite interaction, such as inflammation and soft tissue invasion. Replacement of the single cysteine residue (C-192) with serine (C192S mutation) resulted in loss of detectable proteolytic activity against bovine casein, human fibronectin, and the low-molecular-weight synthetic substrate 7-amino-4-trifluoromethyl coumarin. The C192S mutant molecule does not undergo autocatalytic processing of zymogen to mature form. Taken together, these data support the hypothesis that C-192 participates in active-site formation and enzyme catalysis. PMID:8675287

  12. Substitution of cysteine 192 in a highly conserved Streptococcus pyogenes extracellular cysteine protease (interleukin 1beta convertase) alters proteolytic activity and ablates zymogen processing.

    PubMed

    Musser, J M; Stockbauer, K; Kapur, V; Rudgers, G W

    1996-06-01

    Virtually all strains of the human pathogenic bacterium Streptococcus pyogenes express a highly conserved extracellular cysteine protease. The protein is made as an inactive zymogen of 40,000 Da and undergoes autocatalytic truncation to result in a 28,000-Da active protease. Numerous independent lines of investigation suggest that this enzyme participates in one or more phases of host-parasite interaction, such as inflammation and soft tissue invasion. Replacement of the single cysteine residue (C-192) with serine (C192S mutation) resulted in loss of detectable proteolytic activity against bovine casein, human fibronectin, and the low-molecular-weight synthetic substrate 7-amino-4-trifluoromethyl coumarin. The C192S mutant molecule does not undergo autocatalytic processing of zymogen to mature form. Taken together, these data support the hypothesis that C-192 participates in active-site formation and enzyme catalysis.

  13. Pestivirus gene expression: the first protein product of the bovine viral diarrhea virus large open reading frame, p20, possesses proteolytic activity.

    PubMed Central

    Wiskerchen, M; Belzer, S K; Collett, M S

    1991-01-01

    The positive-strand RNA genome of pestiviruses contains a single large open reading frame (ORF) extending its entire length and is capable of encoding 450 kDa of protein. Studies have been undertaken with the purpose of elucidating the specific mechanisms involved in the biogenesis of the complete complement of pestivirus proteins. Here, we report on gene expression at the 5' end of the genome of the prototype pestivirus, bovine viral diarrhea virus (BVDV). We demonstrate, using both a cell-free transcription-translation system and a mammalian-cell transient-expression system, that the first protein product of the large ORF of BVDV, the p20 protein, possesses a specific proteolytic activity. The p20 proteinase activity acts to release the p20 protein from the nascent polyprotein. The p20 proteinase activity is not, however, required for downstream glycoprotein processing, indicating translocation of the pestivirus glycoprotein precursor is affected by an internal signal sequence. Images PMID:1649345

  14. Proteolytic activity among various oral Treponema species and cloning of a prtP-like gene of Treponema socranskii subsp. socranskii.

    PubMed

    Heuner, K; Bergmann, I; Heckenbach, K; Göbel, U B

    2001-07-24

    The proteolytic activity of 11 treponemal strains representing different phylogenetic groups was investigated by SDS-polyacrylamide gel electrophoresis with copolymerised casein, gelatin and fibrinogen as substrates. The activity was specified to be trypsin- and chymotrypsin-like by the cleavage of synthetic substrates BAPNA and SAAPFNA, respectively. Nine strains degrade casein and the synthetic substrate BAPNA. Chymotrypsin-like activity specifically inhibited by phenylmethylsulfonyl fluoride was found in four treponemes. Southern blot analysis using a Treponema socranskii subsp. socranskii-specific prtP probe confirmed the presence of prtP homologous genes in these four strains. The internal fragments of the chymotrypsin-like protease genes were cloned and sequenced after PCR amplification. Here we report the cloning of the complete prtP-like gene of T. socranskii subsp. socranskii, an organism shown to possess epidemiologic relevance in periodontitis.

  15. Effect of Allium sativum and fish collagen on the proteolytic and angiotensin-I converting enzyme-inhibitory activities in cheese and yogurt.

    PubMed

    Shori, A B; Baba, A S; Keow, J N

    2012-12-15

    There is an increasing demand of functional foods in developed countries. Yogurt plays an important role in the management of blood pressure. Several bioactive peptides isolated from Allium sativum or fish collagen have shown antihypertensive activity. Thus, in the present study the effects of A. sativum and/or Fish Collagen (FC) on proteolysis and ACE inhibitory activity in yogurt (0, 7 and 14 day) and cheese (0, 14 and 28 day) were investigated. Proteolytic activities were the highest on day 7 of refrigerated storage in A. sativum-FC-yogurt (337.0 +/- 5.3 microg g(-1)) followed by FC-yogurt (275.3 +/- 2.0 microg g(-1)), A. sativum-yogurt (245.8 +/- 4.2 microg g(-1)) and plain-yogurt (40.4 +/- 1.2 microg g(-1)). On the other hand, proteolytic activities in cheese ripening were the highest (p < 0.05) on day 14 of storage for plain and A. sativum-cheeses (411.4 +/- 4.3 and 528.7 +/- 1.6 microg g(-1), respectively). However, the presence of FC increased the proteolysis to the highest level on day 28 of storage for FC- and A. sativum-FC cheeses (641.2 +/- 0.1 and 1128.4 +/- 4.5 microg g(-1), respectively). In addition, plain- and A. sativum-yogurts with or without FC showed maximal inhibition of ACE on day 7 of storage. Fresh plain- and A. sativum-cheeses showed ACE inhibition (72.3 +/- 7.8 and 50.4 +/- 1.6 % respectively), the presence of FC in both type of cheeses reduced the ACE inhibition to 62.9 +/- 0.8 and 44.5 +/- 5.0%, respectively. However, refrigerated storage increased ACE inhibition in cheeses (p < 0.05 on day 28) in the presence of FC more than in the absence. In conclusion, the presence of FC in A. sativum-yogurt or cheese enhanced the proteolytic activity. Thus, it has potential in the development of an effective dietary strategy for hypertension associated cardiovascular diseases.

  16. Docosahexaenoic acid inhibits proteolytic processing of sterol regulatory element-binding protein-1c (SREBP-1c) via activation of AMP-activated kinase.

    PubMed

    Deng, Xiong; Dong, Qingming; Bridges, Dave; Raghow, Rajendra; Park, Edwards A; Elam, Marshall B

    2015-12-01

    In hyperinsulinemic states including obesity and T2DM, overproduction of fatty acid and triglyceride contributes to steatosis of the liver, hyperlipidemia and hepatic insulin resistance. This effect is mediated in part by the transcriptional regulator sterol responsive element binding protein-1c (SREBP-1c), which stimulates the expression of genes involved in hepatic fatty acid and triglyceride synthesis. SREBP-1c is up regulated by insulin both via increased transcription of nascent full-length SREBP-1c and by enhanced proteolytic processing of the endoplasmic reticulum (ER)-bound precursor to yield the transcriptionally active n-terminal form, nSREBP-1c. Polyunsaturated fatty acids of marine origin (n-3 PUFA) prevent induction of SREBP-1c by insulin thereby reducing plasma and hepatic triglycerides. Despite widespread use of n-3 PUFA supplements to reduce triglycerides in clinical practice, the exact mechanisms underlying their hypotriglyceridemic effect remain elusive. Here we demonstrate that the n-3 PUFA docosahexaenoic acid (DHA; 22:5 n-3) reduces nSREBP-1c by inhibiting regulated intramembrane proteolysis (RIP) of the nascent SREBP-1c. We further show that this effect of DHA is mediated both via activation of AMP-activated protein kinase (AMPK) and by inhibition of mechanistic target of rapamycin complex 1 (mTORC1). The inhibitory effect of AMPK on SREBP-1c processing is linked to phosphorylation of serine 365 of SREBP-1c in the rat. We have defined a novel regulatory mechanism by which n-3 PUFA inhibit induction of SREBP-1c by insulin. These findings identify AMPK as an important negative regulator of hepatic lipid synthesis and as a potential therapeutic target for hyperlipidemia in obesity and T2DM.

  17. Clonal Evolution and Blast Crisis Correlate with Enhanced Proteolytic Activity of Separase in BCR-ABL b3a2 Fusion Type CML under Imatinib Therapy

    PubMed Central

    Haaß, Wiltrud; Kleiner, Helga; Weiß, Christel; Haferlach, Claudia; Schlegelberger, Brigitte; Müller, Martin C.; Hehlmann, Rüdiger; Hofmann, Wolf-Karsten; Fabarius, Alice; Seifarth, Wolfgang

    2015-01-01

    Unbalanced (major route) additional cytogenetic aberrations (ACA) at diagnosis of chronic myeloid leukemia (CML) indicate an increased risk of progression and shorter survival. Moreover, newly arising ACA under imatinib treatment and clonal evolution are considered features of acceleration and define failure of therapy according to the European LeukemiaNet (ELN) recommendations. On the basis of 1151 Philadelphia chromosome positive chronic phase patients of the randomized CML-study IV, we examined the incidence of newly arising ACA under imatinib treatment with regard to the p210BCR-ABL breakpoint variants b2a2 and b3a2. We found a preferential acquisition of unbalanced ACA in patients with b3a2 vs. b2a2 fusion type (ratio: 6.3 vs. 1.6, p = 0.0246) concurring with a faster progress to blast crisis for b3a2 patients (p = 0.0124). ESPL1/Separase, a cysteine endopeptidase, is a key player in chromosomal segregation during mitosis. Separase overexpression and/or hyperactivity has been reported from a wide range of cancers and cause defective mitotic spindles, chromosome missegregation and aneuploidy. We investigated the influence of p210BCR-ABL breakpoint variants and imatinib treatment on expression and proteolytic activity of Separase as measured with a specific fluorogenic assay on CML cell lines (b2a2: KCL-22, BV-173; b3a2: K562, LAMA-84). Despite a drop in Separase protein levels an up to 5.4-fold increase of Separase activity under imatinib treatment was observed exclusively in b3a2 but not in b2a2 cell lines. Mimicking the influence of imatinib on BV-173 and LAMA-84 cells by ESPL1 silencing stimulated Separase proteolytic activity in both b3a2 and b2a2 cell lines. Our data suggest the existence of a fusion type-related feedback mechanism that posttranslationally stimulates Separase proteolytic activity after therapy-induced decreases in Separase protein levels. This could render b3a2 CML cells more prone to aneuploidy and clonal evolution than b2a2 progenitors

  18. Clonal Evolution and Blast Crisis Correlate with Enhanced Proteolytic Activity of Separase in BCR-ABL b3a2 Fusion Type CML under Imatinib Therapy.

    PubMed

    Haaß, Wiltrud; Kleiner, Helga; Weiß, Christel; Haferlach, Claudia; Schlegelberger, Brigitte; Müller, Martin C; Hehlmann, Rüdiger; Hofmann, Wolf-Karsten; Fabarius, Alice; Seifarth, Wolfgang

    2015-01-01

    Unbalanced (major route) additional cytogenetic aberrations (ACA) at diagnosis of chronic myeloid leukemia (CML) indicate an increased risk of progression and shorter survival. Moreover, newly arising ACA under imatinib treatment and clonal evolution are considered features of acceleration and define failure of therapy according to the European LeukemiaNet (ELN) recommendations. On the basis of 1151 Philadelphia chromosome positive chronic phase patients of the randomized CML-study IV, we examined the incidence of newly arising ACA under imatinib treatment with regard to the p210BCR-ABL breakpoint variants b2a2 and b3a2. We found a preferential acquisition of unbalanced ACA in patients with b3a2 vs. b2a2 fusion type (ratio: 6.3 vs. 1.6, p = 0.0246) concurring with a faster progress to blast crisis for b3a2 patients (p = 0.0124). ESPL1/Separase, a cysteine endopeptidase, is a key player in chromosomal segregation during mitosis. Separase overexpression and/or hyperactivity has been reported from a wide range of cancers and cause defective mitotic spindles, chromosome missegregation and aneuploidy. We investigated the influence of p210BCR-ABL breakpoint variants and imatinib treatment on expression and proteolytic activity of Separase as measured with a specific fluorogenic assay on CML cell lines (b2a2: KCL-22, BV-173; b3a2: K562, LAMA-84). Despite a drop in Separase protein levels an up to 5.4-fold increase of Separase activity under imatinib treatment was observed exclusively in b3a2 but not in b2a2 cell lines. Mimicking the influence of imatinib on BV-173 and LAMA-84 cells by ESPL1 silencing stimulated Separase proteolytic activity in both b3a2 and b2a2 cell lines. Our data suggest the existence of a fusion type-related feedback mechanism that posttranslationally stimulates Separase proteolytic activity after therapy-induced decreases in Separase protein levels. This could render b3a2 CML cells more prone to aneuploidy and clonal evolution than b2a2 progenitors

  19. Angelman syndrome-associated ubiquitin ligase UBE3A/E6AP mutants interfere with the proteolytic activity of the proteasome.

    PubMed

    Tomaić, V; Banks, L

    2015-01-29

    Angelman syndrome, a severe neurodevelopmental disease, occurs primarily due to genetic defects, which cause lack of expression or mutations in the wild-type E6AP/UBE3A protein. A proportion of the Angelman syndrome patients bear UBE3A point mutations, which do not interfere with the expression of the full-length protein, however, these individuals still develop physiological conditions of the disease. Interestingly, most of these mutations are catalytically defective, thereby indicating the importance of UBE3A enzymatic activity role in the Angelman syndrome pathology. In this study, we show that Angelman syndrome-associated mutants interact strongly with the proteasome via the S5a proteasomal subunit, resulting in an overall inhibitory effect on the proteolytic activity of the proteasome. Our results suggest that mutated catalytically inactive forms of UBE3A may cause defects in overall proteasome function, which could have an important role in the Angelman syndrome pathology.

  20. Nitrogen mustard exposure of murine skin induces DNA damage, oxidative stress and activation of MAPK/Akt-AP1 pathway leading to induction of inflammatory and proteolytic mediators.

    PubMed

    Kumar, Dileep; Tewari-Singh, Neera; Agarwal, Chapla; Jain, Anil K; Inturi, Swetha; Kant, Rama; White, Carl W; Agarwal, Rajesh

    2015-06-15

    Our recent studies in SKH-1 hairless mice have demonstrated that topical exposure to nitrogen mustard (NM), an analog of sulfur mustard (SM), triggers the inflammatory response, microvesication and apoptotic cell death. Here, we sought to identify the mechanism/s involved in these NM-induced injury responses. Results obtained show that NM exposure of SKH-1 hairless mouse skin caused H2A.X and p53 phosphorylation and increased p53 accumulation, indicating DNA damage. In addition, NM also induced the activation of MAPKs/ERK1/2, JNK1/2 and p38 as well as that of Akt together with the activation of transcription factor AP1. Also, NM exposure induced robust expression of pro-inflammatory mediators namely cyclooxygenase 2 and inducible nitric oxide synthase and cytokine tumor necrosis factor alpha, and increased the levels of proteolytic mediator matrix metalloproteinase 9. NM exposure of skin also increased lipid peroxidation, 5,5-dimethyl-2-(8-octanoic acid)-1-pyrroline N-oxide protein adduct formation, protein and DNA oxidation indicating an elevated oxidative stress. We also found NM-induced increase in the homologous recombinant repair pathway, suggesting its involvement in the repair of NM-induced DNA damage. Collectively, these results indicate that NM induces oxidative stress, mainly a bi-phasic response in DNA damage and activation of MAPK and Akt pathways, which activate transcription factor AP1 and induce the expression of inflammatory and proteolytic mediators, contributing to the skin injury response by NM. In conclusion, this study for the first time links NM-induced mechanistic changes with our earlier reported murine skin injury lesions with NM, which could be valuable to identify potential therapeutic targets and rescue agents. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  1. Comparison of proteolytic activities produced by entomopathogenic Photorhabdus bacteria: strain- and phase-dependent heterogeneity in composition and activity of four enzymes.

    PubMed

    Marokházi, Judit; Lengyel, Katalin; Pekár, Szilvia; Felföldi, Gabriella; Patthy, András; Gráf, László; Fodor, András; Venekei, István

    2004-12-01

    Twenty strains (including eight phase variant pairs) of nematode-symbiotic and insect-pathogenic Photorhabdus bacteria were examined for the production of proteolytic enzymes by using a combination of several methods, including gelatin liquefaction, zymography coupled to native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and activity measurement with two chromogen substrate types. Four protease activities (approximately 74, approximately 55, approximately 54, and approximately 37 kDa) could be separated. The N-terminal sequences of three of the proteases were determined, and a comparison with sequences in databases allowed identification of these proteases as HEXXH metallopeptidases. Thus, the 74-kDa protease (described formerly as Php-B [J. Marokhazi, G. Koczan, F. Hudecz, L. Graf, A. Fodor, and I. Venekei, Biochem. J. 379:633-640, 2004) is an ortholog of OpdA, a member the thimet oligopeptidase family, and the 55-kDa protease is an ortholog of PrtA, a HEXXH+H peptidase in clan MB (metzincins), while the 37-kDa protease (Php-C) belongs to the HEXXH+E peptidases in clan MA. The 54-kDa protease (Php-D) is a nonmetalloenzyme. PrtA and Php-C were zymographically detected, and they occurred in several smaller forms as well. OpdA could not be detected by zymography. PrtA, Php-C, and Php-D were secreted proteases; OpdA, in contrast, was an intracellular enzyme. OpdA activity was found in every strain tested, while Php-D was detected only in the Brecon/1 strain. There was significant strain variation in the secretion of PrtA and Php-C activities, but reduced activity or a lack of activity was not specific to secondary-phase variants. The presence of PrtA, OpdA, and Php-C activities could be detected in the hemolymph of Galleria melonella larvae 20 to 40 h postinfection. These proteases appear not to be directly involved in the pathogenicity of Photorhabdus, since strains or phase variants lacking any of these proteases do not show reduced virulence when

  2. Effects of Pleurotus eryngii polysaccharides on bacterial growth, texture properties, proteolytic capacity, and angiotensin-I-converting enzyme-inhibitory activities of fermented milk.

    PubMed

    Li, Siqian; Shah, Nagendra P

    2015-05-01

    Pleurotus eryngii is one of the most favored oyster mushrooms and contains various beneficial bioactive compounds. Polysaccharide extracted from P. eryngii (PEPS) was added as a natural-source ingredient to milk before fermentation, and the effects of additional PEPS on fermented milk were investigated in this study. The PEPS were extracted and added to reconstituted skim milk (12%, wt/vol) at 0.5, 0.25, and 0.125% (wt/vol) and fermented by a non-exopolysaccharide-producing strain, Streptococcus thermophilus Australian Starter Culture Collection (ASCC) 1303 (ST 1303), or an exopolysaccharide-producing Strep. thermophilus ASCC 1275 (ST 1275). Bacterial growth, texture properties, microstructure, proteolytic capacity, and angiotensin-I-converting enzyme-inhibitory activities of fermented milk (FM) were determined during refrigerated storage at 4°C for 21d. Viable counts of starter bacteria in FM with 0.5% PEPS added were the highest. Changes in pH were consistent with changes in titratable acidities for all samples. The FM samples with added PEPS showed denser protein aggregates containing larger serum pores in confocal micrographs compared with those without PEPS at d 0 and 21during refrigerated storage. The values for spontaneous whey separation of FM with added PEPS were significantly higher than those of FM fermented by ST 1303 or ST 1275 without PEPS. The proteolytic activities of ST 1303 of FM with added PEPS were higher than those of FM fermented by ST 1303 without PEPS. The FM with added 0.125% PEPS had similar angiotensin-I-converting enzyme-inhibitory activity to that fermented by ST 1303 without PEPS; both were higher than those of other samples during refrigerated storage. Firmness and gumminess values of FM with added PEPS were higher than those of FM fermented by ST 1303 or ST 1275 without PEPS. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  3. Responses of black and white skin to solar-simulating radiation: differences in DNA photodamage, infiltrating neutrophils, proteolytic enzymes induced, keratinocyte activation, and IL-10 expression.

    PubMed

    Rijken, Feiko; Bruijnzeel, Piet L B; van Weelden, Huib; Kiekens, Rebecca C M

    2004-06-01

    Black skin is more resistant to the deleterious effects of ultraviolet radiation than white skin. A higher melanin content and a different melanosomal dispersion pattern in the epidermis are thought to be responsible for this. Our purpose was to compare skin responses in black and white skin following exposure to solar-simulating radiation (SSR) to further investigate the photoprotective properties of black skin. Six volunteers of skin phototype I-III (white) were exposed to (doses measured directly with a Waldmann UV detector device) 12,000-18,000 mJ per cm2 (2 MED) of SSR and compared with six volunteers of skin phototype VI (black) exposed to 18,000 mJ per cm2 (<1 MED) of SSR. The presence and distribution of skin pigment, DNA photodamage, infiltrating neutrophils, photoaging associated proteolytic enzymes, keratinocyte activation, and the source of interleukin 10 (IL-10) in skin biopsies taken before and after exposure were studied. In all white skinned subjects, 12,000-18,000 mJ per cm2 of SSR induced DNA damage in epidermal and dermal cells, an influx of neutrophils, active proteolytic enzymes, and diffuse keratinocyte activation. Additionally, in three of the white skinned volunteers IL-10 positive neutrophils were found to infiltrate the epidermis. Except for DNA damage in the supra basal epidermis, none of these changes was found in black skinned subjects. Increased skin pigmentation appears to be primarily responsible for the observed differences in skin responses. Our data could provide an explanation as to why black skin is less susceptible to sunburn, photoaging, and skin carcinogenesis.

  4. Trafficking and Proteolytic Processing of APP

    PubMed Central

    Haass, Christian; Kaether, Christoph; Thinakaran, Gopal; Sisodia, Sangram

    2012-01-01

    Accumulations of insoluble deposits of amyloid β-peptide are major pathological hallmarks of Alzheimer disease. Amyloid β-peptide is derived by sequential proteolytic processing from a large type I trans-membrane protein, the β-amyloid precursor protein. The proteolytic enzymes involved in its processing are named secretases. β- and γ-secretase liberate by sequential cleavage the neurotoxic amyloid β-peptide, whereas α-secretase prevents its generation by cleaving within the middle of the amyloid domain. In this chapter we describe the cell biological and biochemical characteristics of the three secretase activities involved in the proteolytic processing of the precursor protein. In addition we outline how the precursor protein maturates and traffics through the secretory pathway to reach the subcellular locations where the individual secretases are preferentially active. Furthermore, we illuminate how neuronal activity and mutations which cause familial Alzheimer disease affect amyloid β-peptide generation and therefore disease onset and progression. PMID:22553493

  5. Human pregnancy serum contains at least two distinct proteolytic activities with the ability to degrade insulin-like growth factor binding protein-3.

    PubMed

    Bang, P; Fielder, P J

    1997-09-01

    The presence of a proteolytic activity in sera from pregnant humans and rodents capable of degrading insulin-like growth factor binding protein-3 (IGFBP-3) has been known for some time. However, the identity of this activity has remained elusive. We have attempted to purify the IGFBP-3 protease activity from pregnant human serum (PHS) using the degradation of 125I-IGFBP-3 as a marker. Following ammonium sulfate precipitation of PHS and further enrichment of active fractions by ion-exchange, protein-A Sepharose, and size-exclusion chromatography, a protease of approximately 70-90 kDa was isolated and subjected to N-terminal analysis. The N-terminal sequence was consistent with plasminogen, a known fibrinolytic enzyme. To further characterize the IGFBP-3 protease activities in both PHS and nonpregnant human serum (NHS), aliquots of serum were first enriched by polyethylene glycol-precipitation and subjected to size-exclusion chromatography. The size-separated fractions were then incubated with 125I-IGFBP-3, and proteolytic activity was measured. PHS contained two separate proteases (>150 kDa and 70-90 kDa), whereas NHS contained only one (70-90 kDa) that had a inhibitor profile similar to plasmin. However, inhibitors of plasmin had no effect on the activity of the >150-kDa protease. Plasminogen activators (PAs) greatly increased the activity of the 70- to 90-kDa protease, but had little effect on the >150-kDa protease activity. Addition of PAs greatly increased the ability of NHS to proteolyze IGFBP-3. In contrast, the ability of plasminogen-depleted plasma to degrade 125I-IGFBP-3 was not affected by the addition of PAs. Both urokinase and tissue-type PA had the ability to proteolyze IGFBP-3 and were, in contrast to the >150-kDa protease activity, inhibited by the specific PA inhibitor D-PHE-PRO-ARG chloromethyl ketone. The present data suggest that sera has the ability to proteolyze IGFBP-3, and that this ability, as demonstrated by NHS, can be regulated by protease

  6. Protease analysis by neoepitope approach reveals the activation of MMP-9 is achieved proteolytically in a test tissue cartilage model involved in bone formation.

    PubMed

    Lee, Eunice R; Lamplugh, Lisa; Kluczyk, Beata; Mort, John S; Leblond, Charles Philippe

    2006-09-01

    A principle of regulation of matrix metalloproteinase (MMP) activity has been introduced as the cysteine-switch mechanism of activation (Springman et al. 1990). According to this mechanism, a critical Cys residue found in the auto-inhibitory propeptide domain of latent proenzyme is important to determine whether or not activation is turned on or off. The mechanism further allows for multiple modes of activation. To determine whether or not activation is accomplished proteolytically within a rat test cartilage model, protease analysis by the neoepitope approach, which relies upon a set of antibodies, was applied. One is used to identify the MMP-9 proenzyme bearing the critical cysteine residue, the other to identify any enzyme present bearing a new NH2-terminus 89FQTFD. This is indicative of MMP-9 lacking the cysteine switch. The antibody set has been applied to frozen tissue sections and analyzed by light and electron microscopic methods. Results reveal that activation of the MMP-9 protease involves limited proteolysis resulting in propeptide domain release. Here we report the observed changes of protease form to indigenous cells and extracellular matrix, thereby making it possible to uncover the features of MMP-9 activation within a specified set of tissue circumstances where a cartilage model is transformed into definitive bone. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

  7. Kallikrein-related Peptidase-8 (KLK8) Is an Active Serine Protease in Human Epidermis and Sweat and Is Involved in a Skin Barrier Proteolytic Cascade

    PubMed Central

    Eissa, Azza; Amodeo, Vanessa; Smith, Christopher R.; Diamandis, Eleftherios P.

    2011-01-01

    Kallikrein-related peptidase-8 (KLK8) is a relatively uncharacterized epidermal protease. Although proposed to regulate skin-barrier desquamation and recovery, the catalytic activity of KLK8 was never demonstrated in human epidermis, and its regulators and targets remain unknown. Herein, we elucidated for the first time KLK8 activity in human non-palmoplantar stratum corneum and sweat ex vivo. The majority of stratum corneum and sweat KLK8 was catalytically active, displaying optimal activity at pH 8.5 and considerable activity at pH 5. We also showed that KLK8 is a keratinocyte-specific protease, not secreted by human melanocytes or dermal fibroblasts. KLK8 secretion increased significantly upon calcium induction of terminal keratinocyte differentiation, suggesting an active role for this protease in upper epidermis. Potential activators, regulators, and targets of KLK8 activity were identified by in vitro kinetic assays using pro-KLK8 and mature KLK8 recombinant proteins produced in Pichia pastoris. Mature KLK8 activity was enhanced by calcium and magnesium ions and attenuated by zinc ions and by autocleavage after Arg164. Upon screening KLK8 cleavage of a library of FRET-quenched peptides, trypsin-like specificity was observed with the highest preference for (R/K)(S/T)(A/V) at P1-P1′-P2′. We also demonstrated that KLK5 and lysyl endopeptidase activate latent pro-KLK8, whereas active KLK8 targets pro-KLK11, pro-KLK1, and LL-37 antimicrobial peptide activation in vitro. Together, our data identify KLK8 as a new active serine protease in human stratum corneum and sweat, and we propose regulators and targets that augment its involvement in a skin barrier proteolytic cascade. The implications of KLK8 elevation and hyperactivity in desquamatory and inflammatory skin disease conditions remain to be studied. PMID:20940292

  8. Impact of microbial growth inhibition and proteolytic activity on the stability of a new formulation containing a phytate-degrading enzyme obtained from mushroom.

    PubMed

    Spier, Michele R; Siepmann, Francieli B; Staack, Larissa; Souza, Priscila Z; Kumar, Vikas; Medeiros, Adriane B P; Soccol, Carlos R

    2016-10-02

    The development of stable enzymes is a key issue in both the food and feed industries. Consequently, the aim of the current study is to evaluate the impact of various additives (sodium chloride, sodium citrate, mannitol, methylparaben, polyethylene glycol 3350, ethylenediaminetetraacetic acid disodium salt, and a serine protease inhibitor) on the stability of a mushroom phytase produced by solid-state cultivation and recovery. Also observed was the effect of the additives on microbial growth inhibition by monitoring both the change in optical density over 30 days of storage and proteolytic activity. Initially, eight experimental formulations were prepared along with a control. After screening, a 3(2) factorial design was applied to define suitable concentrations of the selected additives. Among the eight formulations tested, the formulation containing NaCl, PEG 3350, and methylparaben retained all of the initial phytase activity after 50 days of storage, with no detected interference from protease activity. Sodium citrate, a metal chelation agent, presented the unusual effect of reducing protease activity in the formulations. Although all formulations presented better phytase stability when compared to the control, NaCl and PEG were both able to prolong the stability of the enzyme activity and also to inhibit microbial growth during storage, making them favorable for application as food and feed additives.

  9. Betulin suppressed interleukin-1β-induced gene expression, secretion and proteolytic activity of matrix metalloproteinase in cultured articular chondrocytes and production of matrix metalloproteinase in the knee joint of rat

    PubMed Central

    Ra, Ho Jong; Lee, Hyun Jae; Jo, Ho Seung; Nam, Dae Cheol; Lee, Young Bok; Kang, Byeong Hun; Moon, Dong Kyu; Kim, Dong Hee

    2017-01-01

    We investigated whether betulin affects the gene expression, secretion and proteolytic activity of matrix metalloproteinase-3 (MMP-3) in primary cultured rabbit articular chondrocytes, as well as in vivo production of MMP-3 in the rat knee joint to evaluate the potential chondroprotective effect of betulin. Rabbit articular chondrocytes were cultured and reverse transcription-polymerase chain reaction (RT-PCR) was used to measure interleukin-1β (IL-1β)-induced gene expression of MMP-3, MMP-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), ADAMTS-5 and type II collagen. Effect of betulin on IL-1β-induced secretion and proteolytic activity of MMP-3 was investigated using western blot analysis and casein zymography, respectively. Effect of betulin on MMP-3 protein production was also examined in vivo. The results were as follows: (1) betulin inhibited the gene expression of MMP-3, MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5, but increased the gene expression of type II collagen; (2) betulin inhibited the secretion and proteolytic activity of MMP-3; (3) betulin suppressed the production of MMP-3 protein in vivo. These results suggest that betulin can regulate the gene expression, secretion, and proteolytic activity of MMP-3, by directly acting on articular chondrocytes. PMID:28066137

  10. Betulin suppressed interleukin-1β-induced gene expression, secretion and proteolytic activity of matrix metalloproteinase in cultured articular chondrocytes and production of matrix metalloproteinase in the knee joint of rat.

    PubMed

    Ra, Ho Jong; Lee, Hyun Jae; Jo, Ho Seung; Nam, Dae Cheol; Lee, Young Bok; Kang, Byeong Hun; Moon, Dong Kyu; Kim, Dong Hee; Lee, Choong Jae; Hwang, Sun-Chul

    2017-01-01

    We investigated whether betulin affects the gene expression, secretion and proteolytic activity of matrix metalloproteinase-3 (MMP-3) in primary cultured rabbit articular chondrocytes, as well as in vivo production of MMP-3 in the rat knee joint to evaluate the potential chondroprotective effect of betulin. Rabbit articular chondrocytes were cultured and reverse transcription-polymerase chain reaction (RT-PCR) was used to measure interleukin-1β (IL-1β)-induced gene expression of MMP-3, MMP-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), ADAMTS-5 and type II collagen. Effect of betulin on IL-1β-induced secretion and proteolytic activity of MMP-3 was investigated using western blot analysis and casein zymography, respectively. Effect of betulin on MMP-3 protein production was also examined in vivo. The results were as follows: (1) betulin inhibited the gene expression of MMP-3, MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5, but increased the gene expression of type II collagen; (2) betulin inhibited the secretion and proteolytic activity of MMP-3; (3) betulin suppressed the production of MMP-3 protein in vivo. These results suggest that betulin can regulate the gene expression, secretion, and proteolytic activity of MMP-3, by directly acting on articular chondrocytes.

  11. Neutrophil proteinase cathepsin G is proteolytically active on the human platelet glycoprotein Ib-IX receptor: characterization of the cleavage sites within the glycoprotein Ib alpha subunit.

    PubMed Central

    Pidard, D; Renesto, P; Berndt, M C; Rabhi, S; Clemetson, K J; Chignard, M

    1994-01-01

    The proteolytic activity of the neutrophil serine-proteinase cathepsin G (CG) on platelet adherence receptors, the glycoprotein (GP) Ib-IX complex and the integrin alpha IIb beta 3, has been investigated. In the range 50 to 200 nmol/l, CG is a potent platelet agonist which induces shape change, granule exocytosis and aggregation. Investigation of the proteolysis of the receptors' subunits during the course of platelet activation by CG was performed by immunoblot analysis of platelet proteins using a panel of specific antibodies. Exposure of platelets for 3 min at 37 degrees C to CG at a concentration that induces full cell activation resulted in an extensive cleavage of the N-terminal region of the extracellular domain of GPIb alpha, the largest (relative molecular mass, M(r), 143,000) of the three subunits constituting the GPIb-IX complex. In contrast, no detectable proteolytic modification of the two other subunits, GPIb beta and GPIX, was detected. Similarly, we observed that neither of the two subunits of the alpha IIb beta 3 receptor were proteolytically modified by CG. Cleavage of GPIb alpha by CG leaves a remnant of the polypeptide chain with M(r) approx. 106,000 in the plasma membrane, while releasing into the extracellular milieu the N-terminal domain with M(r) in the range 40,000 to 46,000. N-terminal sequencing of the CG-derived fragments of GPIb alpha indicated that the Leu275-Tyr276 peptide bond was the primary cleavage site for this proteinase. Proteolysis of GPIb alpha was already detectable at concentrations of CG as low as 25 nmol/l, while with 200 nmol/l the cleavage was detected as soon as 10 s after exposure of platelets to the proteinase. Comparison of the kinetics and concentration dependency for the proteolysis of GPIb alpha and for the activation of platelets by CG showed that cleavage of the GPIb-IX receptor is an early event that accompanies exocytosis and aggregation. Quantitative evaluation of the conversion of GPIb alpha into its

  12. A Membrane Protease Regulates Energy Production in Macrophages by Activating Hypoxia-inducible Factor-1 via a Non-proteolytic Mechanism*

    PubMed Central

    Sakamoto, Takeharu; Seiki, Motoharu

    2010-01-01

    Most cells produce ATP in the mitochondria by oxidative phosphorylation. However, macrophages, which are major players in the innate immune system, use aerobic glycolysis to produce ATP. HIF-1 (hypoxia-inducible factor-1) regulates expression of glycolysis-related genes and maintains macrophage glycolytic activity. However, it is unclear how HIF-1 activity is maintained in macrophages during normoxia. In this study, we found that macrophages lacking membrane type 1 matrix metalloproteinase (MT1-MMP/MMP-14), a potent invasion-promoting protease, exhibited considerably lower ATP levels than wild-type cells. HIF-1 was activated by an unanticipated function of MT1-MMP, which led to the stimulation of ATP production via glycolysis. The cytoplasmic tail of MT1-MMP bound to FIH-1 (factor inhibiting HIF-1), which led to the inhibition of the latter by its recently identified inhibitor, Mint3/APBA3. We have thus identified a new function of MT1-MMP to mediate production of ATP so as to support energy-dependent macrophage functions by a previously unknown non-proteolytic mechanism. PMID:20663879

  13. Planar integrated optical waveguide used as a transducer to yield chemical information: detection of the activity of proteolytic enzymes e.g. serine-proteases

    NASA Astrophysics Data System (ADS)

    Zhylyak, Gleb; Ramoz-Perez, Victor; Linnhoff, Michael; Hug, Thomas; Citterio, Daniel; Spichiger-Keller, Ursula E.

    2005-03-01

    The paper shows the very first results of a feasibility study where the activity of proteolytic enzymes towards dye-labelled artificial substrates immobilized on the surface of planar optical Ta2O5 waveguide was investigated. Within this project, a chromophore label was developed, synthesized and attached to the carboxy-terminus of specific tripeptides. The goal was to develop a highly sensitive optical assay in order to monitor the activity of serine-proteases by cleavage of the amide bond between peptide and chromophore. On the one hand, a strategy was developed to immobilize the labeled tripeptide unto integrated planar waveguides. On the other hand, an instrument, the so-called "chip-reader" was developed to detect the biological process on the surface of the integrated planar optical waveguide. Surface characteristics were analyzed by XPS, TOF-SIMS and contact angle measurements. A comparison between the effectivity of ATR-photometry on chip using TE0 mode and photometry in transmission mode is discussed.

  14. Apoptosis-related deregulation of proteolytic activities and high serum levels of circulating nucleosomes and DNA in blood correlate with breast cancer progression.

    PubMed

    Roth, Carina; Pantel, Klaus; Müller, Volkmar; Rack, Brigitte; Kasimir-Bauer, Sabine; Janni, Wolfgang; Schwarzenbach, Heidi

    2011-01-06

    As cell-free circulating DNA exists predominantly as mono- and oligonucleosomes, the focus of the current study was to examine the interplay of circulating nucleosomes, DNA, proteases and caspases in blood of patients with benign and malignant breast diseases. The concentrations of cell-free DNA and nucleosomes as well as the protease and caspase activities were measured in serum of patients with benign breast disease (n = 20), primary breast cancer (M0, n = 31), metastatic breast cancer (M1, n = 32), and healthy individuals (n = 28) by PicoGreen, Cell Death Detection ELISA, Protease Fluorescent Detection Kit and Caspase-Glo®3/7 Assay, respectively. Patients with benign and malignant tumors had significantly higher levels of circulating nucleic acids in their blood than healthy individuals (p = 0.001, p = 0.0001), whereas these levels could not discriminate between benign and malignant lesions. Our analyses of all serum samples revealed significant correlations of circulating nucleosome with DNA concentrations (p = 0.001), nucleosome concentrations with caspase activities (p = 0.008), and caspase with protease activities (p = 0.0001). High serum levels of protease and caspase activities associated with advanced tumor stages (p = 0.009). Patients with lymph node-positive breast cancer had significantly higher nucleosome levels in their blood than node-negative patients (p = 0.004). The presence of distant metastases associated with a significant increase in serum nucleosome (p = 0.01) and DNA levels (p = 0.04), and protease activities (p = 0.008). Our findings demonstrate that high circulating nucleic acid concentrations in blood are no indicators of a malignant breast tumor. However, the observed changes in apoptosis-related deregulation of proteolytic activities along with the elevated serum levels of nucleosomes and DNA in blood are linked to breast cancer progression.

  15. Identification of antihyperuricemic peptides in the proteolytic digest of shark cartilage water extract using in vivo activity-guided fractionation.

    PubMed

    Murota, Itsuki; Taguchi, Satoko; Sato, Nobuyuki; Park, Eun Young; Nakamura, Yasushi; Sato, Kenji

    2014-03-19

    A peptide that exerts antihyperuricemic activity after oral administration was identified from a microbial protease (alcalase) digest of the water extract of shark cartilage by in vivo activity-guided fractionation, using oxonate-induced hyperuricemic rats. Water extract of shark cartilage was first fractionated by preparative ampholine-free isoelectric focusing, followed by preparative reversed-phase liquid chromatography. The antihyperuricemic activity of the alcalse digests of the obtained fractions was evaluated using an animal model. Alcalase digests of the basic and hydrophobic fractions exerted antihyperuricemic activity. A total of 18 peptides were identified in the alcalase digest of the final active fraction. These peptides were chemically synthesized and evaluated for antihyperuricemic activity. Tyr-Leu-Asp-Asn-Tyr and Ser-Pro-Pro-Tyr-Trp-Pro-Tyr lowered the serum uric acid level via intravenous injection at 5 mg/kg of body weight. Furthermore, orally administered Tyr-Leu-Asp-Asn-Tyr showed antihyperuricemic activity. Therefore, these peptides are at least partially responsible for the antihyperuricemic activity of the alcalase digest of shark cartilage.

  16. Proteolytically Stable Foldamer Mimics of Host-Defense Peptides with Protective Activities in a Murine Model of Bacterial Infection.

    PubMed

    Teyssières, Emilie; Corre, Jean-Philippe; Antunes, Stephanie; Rougeot, Catherine; Dugave, Christophe; Jouvion, Grégory; Claudon, Paul; Mikaty, Guillain; Douat, Céline; Goossens, Pierre L; Guichard, Gilles

    2016-09-22

    The synthesis of bioinspired unnatural backbones leading to foldamers can provide effective peptide mimics with improved properties in a physiological environment. This approach has been applied to the design of structural mimics of membrane active antimicrobial peptides (AMPs) for which activities in vitro have been reported. Yet activities and pharmacokinetic properties in vivo in animal models have remained largely unexplored. Here, we report helical oligourea AMP mimics that are active in vitro against bacterial forms of Bacillus anthracis encountered in vivo, as well as in vivo in inhalational and cutaneous mouse models of B. anthracis infection. The pharmacokinetic profile and the tissue distribution were investigated by β-radio imager whole-body mapping in mice. Low excretion and recovery of the native oligourea in the kidney following intravenous injection is consistent with high stability in vivo. Overall these results provide useful information that support future biomedical development of urea-based foldamer peptide mimics.

  17. Effects of pre-slaughter stress on proteolytic enzyme activities and muscle quality of farmed Atlantic cod (Gadus morhua).

    PubMed

    Hultmann, Lisbeth; Phu, Tran Minh; Tobiassen, Torbjørn; Aas-Hansen, Øyvind; Rustad, Turid

    2012-10-01

    Farmed Atlantic cod were subjected to a combination of stressors in a holding tank before being killed, pre rigor filleted and stored in ice. At slaughter, a higher level of stress was confirmed by blood physiology analyses. This was further associated with significantly reduced muscle pH and somewhat elevated muscle collagenase-like activity in the stressed fish, whereas no differences in cathepsin-like activities were found. After 5 days of iced storage, the stressed fish had significantly lower water holding capacity, reduced hardness and yellowish colour compared to the control group, and no differences in the other parameters investigated. Independent of pre-slaughter stress, the activities of cathepsin B- and B/L-like enzymes increased and activities of cathepsin D/E- and collagenase-like enzymes decreased with storage.

  18. PA28, an activator of the 20 S proteasome, is inactivated by proteolytic modification at its carboxyl terminus.

    PubMed

    Ma, C P; Willy, P J; Slaughter, C A; DeMartino, G N

    1993-10-25

    PA28, a protein activator of the 20 S proteasome, was previously identified in soluble extracts of bovine red blood cells (Ma, C.-P., Slaughter, C. A., and DeMartino, G. N. (1992) J. Biol. Chem. 267, 10515-10523). To determine whether this regulatory protein is as widely distributed as the proteasome, PA28 content and activity were examined in various eukaryotic tissues by immunoblot analysis and by functional assays of tissue extracts. PA28 protein was present in all sources examined. PA28 activity, however, was not detected in many of these sources, including those with the highest level of PA28 protein. To determine the biochemical basis of this result, PA28 was purified from extracts of rat liver, which had high levels of PA28 protein but no PA28 activity. The resulting purified PA28 had no detectable activity but had native and subunit molecular weights indistinguishable from the active PA28 of bovine red blood cells. Using the inactivation of purified PA28 as an assay, a protein that inactivated PA28 without altering its apparent molecular weight on SDS-polyacrylamide gel electrophoresis was identified, purified, and characterized from bovine liver. It had biochemical and catalytic characteristics similar to those of lysosomal carboxypeptidase B. When leupeptin, an inhibitor of lysosomal carboxypeptidase B, was included in the buffers used for the preparation of PA28, PA28 activity was detected in tissues which otherwise failed to demonstrate this activity. A similar result was obtained when extracts were prepared in a manner that minimized disruption of lysosomes. Other carboxypeptidases such as carboxypeptidase Y and pancreatic carboxypeptidase B also inactivated PA28 without altering its apparent molecular weight. Active PA28 binds to the proteasome to form a protease-activator complex that can be isolated after velocity sedimentation centrifugation through glycerol density gradients. Carboxypeptidase-inactivated PA28 failed to form such a complex

  19. Desiccation tolerance of the resurrection plant Ramonda serbica is associated with dehydration-dependent changes in levels of proteolytic activities.

    PubMed

    Kidrič, Marjetka; Sabotič, Jerica; Stevanović, Branka

    2014-07-15

    The unique response of desiccation-tolerant, or resurrection plants, to extreme drought is accompanied by major changes in the protein pool, raising the possibility of the involvement of proteases. We detected and characterized proteases present in their active state in leaf extracts of desiccated Ramonda serbica Panč., a resurrection plant from the Balkan Peninsula. Plants desiccated under laboratory conditions and maintained in anhydrobiosis for 4 and 14 months revived upon rehydration. Protease activities were determined spectrophotometrically in solution and by zymography on gels. Several endo- and aminopeptidases were detected and characterized by their pH profiles. Their enzyme class was determined using specific inhibitors. Those with higher activities were a serine endopeptidase active against Bz-Arg-pNA with a pH optimum around 9, and aminopeptidases optimally active at pHs from 7 to 9 against Leu-pNA, Met-pNA, Phe-pNA, Pro-pNA and Ala-pNA. The levels of their activities in leaf extracts from desiccated plants were significantly higher than those from rehydrated plants and from regularly watered plants, implying their involvement in the recovery of vegetative tissues from desiccation.

  20. Maturational changes in motility, acrosomal proteolytic activity, and penetrability of the inner perivitelline layer of fowl sperm, during their passage through the male genital tract.

    PubMed

    Ahammad, Muslah U; Nishino, C; Tatemoto, H; Okura, N; Kawamoto, Y; Okamoto, S; Nakada, T

    2011-10-01

    The objective was to examine, in vitro, the motility, acrosomal proteolytic activity (APA), and penetrating ability of fowl sperm recovered from the testis and epididymis, as well as the proximal, middle, and distal vas deferens, to assess the potential fertilizing ability of sperm as a function of maturation. A motile sperm separation technique was used to estimate sperm motility with Accudenz, a gelatin slide technique was used to measure the diameter of the halo around the acrosome of individual sperm as an indication of APA, and a sperm-inner perivitelline layer (IPL) interaction assay was done to estimate the number of hole formations as an indication of sperm penetration into the IPL. Sperm in the testis exhibited the least motility, produced the smallest halos, and created the least number of holes per 0.25 mm(2). Motility, diameter of the halo, and number of holes increased gradually (P < 0.05) from the epididymis to the distal vas deferens and were markedly different (P < 0.05) between testicular and deferent duct sperm. Based on these in vitro experimental findings, we inferred that fowl sperm undergo a gradual process of maturational changes in motility, APA, and penetrability as a means of acquiring potential fertility during their passage throughout the male genital tract. Copyright © 2011 Elsevier Inc. All rights reserved.

  1. Proteolytic processing of the Saccharomyces cerevisiae cell wall protein Scw4 regulates its activity and influences its covalent binding to glucan.

    PubMed

    Grbavac, Antonija; Čanak, Iva; Stuparević, Igor; Teparić, Renata; Mrša, Vladimir

    2017-03-01

    Yeast cell wall contains a number of proteins that are either non-covalently (Scw-proteins), or covalently (Ccw-proteins) bound to β-1,3-glucan, the latter either through GPI-anchors and β-1,6-glucan, or by alkali labile ester linkages between γ-carboxyl groups of glutamic acid and hydroxyl groups of glucoses (Pir-proteins). It was shown that a part of Scw4, previously identified among the non-covalently bound cell wall proteins, was covalently attached to wall polysaccharides by a so far unknown alkali sensitive linkage. Thus Scw4 could be released from cell walls by treatments with hot SDS, mild alkali, or β-1,3-glucanases, respectively. It was further shown that non-covalently bound Scw4 (SDS released) underwent the Kex2 proteolytic processing. In this paper it was demonstrated that Scw4 was also processed by yapsins at a position 9 amino acids downstream of the Kex2 cleavage site. Scw4 cleaved at the yapsin site had a markedly lower potential for covalent attachment to glucan. The overproduction of the fully processed form of Scw4 lead to high mortality, particularly in the stationary phase of growth, and to markedly increased cell size. On the other hand, the overproduction of Scw4 processed only by Kex2 or not processed at all had no apparent change in mortality indicating that only the smallest, completely mature form of Scw4 had the activity leading to observed phenotype changes.

  2. Proteolytic cleavage of AMPKα and intracellular MMP9 expression are both required for TLR4-mediated mTORC1 activation and HIF-1α expression in leukocytes

    PubMed Central

    Zhang, Zhiyong; Amorosa, Louis F.; Coyle, Susette M.; Macor, Marie A.; Lubitz, Sara E.; Carson, Jeffrey L.; Birnbaum, Morris J.; Lee, Leonard Y.; Haimovich, Beatrice

    2015-01-01

    LPS-induced TLR4 activation alters cellular bioenergetics and triggers proteolytic cleavage of AMPKα and HIF-1α expression in leukocytes. In human leukocytes, and more specifically neutrophils, AMPKα cleavage yields 55- and 35-kD protein fragments. Here we address the mechanism by which AMPKα is cleaved, and it’s relevance to human health. Our data indicate that AMPKα cleavage is linked to matrix metalloproteinase (MMP9) expression and that both are required for mTORC1 and S6K1 activation, and HIF-1α expression, in LPS stimulated human and mice leukocytes. Three key observations support this conclusion. First, no changes in AMPKα and TLR4 signaling intermediates (mTORC1/S6K1/ HIF-1α) were detected in LPS-stimulated MMP9-deficient mice leukocytes. Second, recombinant MMP9 cleaved human AMPKα ex vivo, producing degradation products similar in size to those detected following LPS-stimulation. Third, MMP9 inhibitors prevented AMPKα degradation and HIF-1α expression in LPS-activated human leukocytes, while AMPK activators blocked MMP9 and HIF-1α expression. Significantly, AMPKα degradation, MMP9, and TLR4 signaling intermediates were all detected in leukocytes from Type 2 diabetes mellitus (T2DM) patients and patients following cardiopulmonary bypass (CPB) surgery. Plasma from these two patient cohorts induced AMPKα cleavage and TLR4 signaling intermediates in healthy donor leukocytes and either a TLR4 inhibitor or polymyxin prevented these outcomes. Detection of AMPKα degradation, MMP9 expression, and TLR4 signaling intermediates described herein in leukocytes, the most readily available human cells for clinical investigation, may provide a powerful tool for further exploring the role of TLR4 signaling in human diseases, and lead to identification of new, context-specific therapeutic modalities for precision medicine. PMID:26232429

  3. Detection of botulinum neurotoxin serotype A, B, and F proteolytic activity in complex matrices with picomolar to femtomolar sensitivity

    USDA-ARS?s Scientific Manuscript database

    Rapid, high-throughput assays that detect and quantify botulinum neurotoxin (BoNT) activity in diverse matrices are required for environmental, clinical, pharmaceutical, and food testing. The current standard, the mouse bioassay, is sensitive but is low in throughput and accuracy. In this study, we...

  4. The Zymogen-Enteropeptidase System: A Practical Approach to Study the Regulation of Enzyme Activity by Proteolytic Cleavage

    ERIC Educational Resources Information Center

    Pizauro, Joao M., Jr.; Ferro, Jesus A.; de Lima, Andrea C. F.; Routman, Karina S.; Portella, Maria Celia

    2004-01-01

    The present research describes an efficient procedure to obtain high levels of trypsinogen and chymotrypsinogen by using a simple, rapid, and easily reproducible method. The extraction process and the time-course of activation of zymogens can be carried out in a single laboratory period, without sophisticated equipment. The main objective was to…

  5. The Zymogen-Enteropeptidase System: A Practical Approach to Study the Regulation of Enzyme Activity by Proteolytic Cleavage

    ERIC Educational Resources Information Center

    Pizauro, Joao M., Jr.; Ferro, Jesus A.; de Lima, Andrea C. F.; Routman, Karina S.; Portella, Maria Celia

    2004-01-01

    The present research describes an efficient procedure to obtain high levels of trypsinogen and chymotrypsinogen by using a simple, rapid, and easily reproducible method. The extraction process and the time-course of activation of zymogens can be carried out in a single laboratory period, without sophisticated equipment. The main objective was to…

  6. Proteomic analysis and purification of an unusual germin-like protein with proteolytic activity in the latex of Thevetia peruviana.

    PubMed

    de Freitas, Cleverson D T; da Cruz, Wallace T; Silva, Maria Z R; Vasconcelos, Ilka M; Moreno, Frederico B M B; Moreira, Renato A; Monteiro-Moreira, Ana C O; Alencar, Luciana M R; Sousa, Jeanlex S; Rocha, Bruno A M; Ramos, Márcio V

    2016-05-01

    The latex from Thevetia peruviana is rich in plant defense proteins, including a 120 kDa cysteine peptidase with structural characteristics similar to germin-like proteins. More than 20,000 plant species produce latex, including Apocynaceae, Sapotaceae, Papaveraceae and Euphorbiaceae. To better understand the physiological role played by latex fluids, a proteomic analysis of Thevetia peruviana (Pers.) Schum latex was performed using two-dimensional gel electrophoresis and mass spectrometry. A total of 33 proteins (86 %) were identified, including storage proteins, a peptidase inhibitor, cysteine peptidases, peroxidases and osmotins. An unusual cysteine peptidase, termed peruvianin-I, was purified from the latex by a single chromatographic step involving gel filtration. The enzyme (glycoprotein) was inhibited by E-64 and iodoacetamide and exhibited high specific activity towards azocasein (K m 17.6 µM), with an optimal pH and temperature of 5.0-6.0 and 25-37 °C, respectively. Gel filtration chromatography, two-dimensional gel electrophoresis, and mass spectrometry revealed that peruvianin-I possesses 120 kDa, pI 4.0, and six subunits (20 kDa). A unique N-terminal amino acid sequence was obtained to oligomer and monomers of peruvianin-I (1ADPGPLQDFCLADLNSPLFINGYPCRNPALAISDDF36). High-resolution images from atomic force microscopy showed the homohexameric structure of peruvianin-I may be organized as a trimer of dimers that form a central channel similar to germin-like proteins. Peruvianin-I exhibited no oxalate oxidase and superoxide dismutase activity or antifungal effects. Peruvianin-I represents the first germin-like protein (GLP) with cysteine peptidase activity, an activity unknown in the GLP family so far.

  7. X-ray crystal structure of CMS1MS2: a high proteolytic activity cysteine proteinase from Carica candamarcensis.

    PubMed

    Gomes, Marco T R; Teixeira, Raphael D; Lopes, Míriam T P; Nagem, Ronaldo A P; Salas, Carlos E

    2012-12-01

    CMS1MS2 (CC-Ib) from Carica candamarcensis (Vasconcellea cundinamarcensis) is a cysteine proteinase found as a single polypeptide containing 213 residues of 22,991 Da. The enzyme was purified by three chromatographic steps, two of them involving cationic exchange. Crystals of CMS1MS2 complexed with E-64 were obtained by the hanging drop vapor-diffusion method at 291 K using ammonium sulfate and polyethylene glycol 4000/8000 as precipitant. The complex CMS1MS2-E-64 crystallized in the tetragonal space group P4(1)2(1)2 with unit-cell parameters; a = b = 73.64, c = 118.79 Å. The structure was determined by Molecular Replacement and refined at 1.87 Å resolution to a final R factor of 16.2 % (R (free) = 19.3 %). Based on the model, the structure of CMS1MS2 (PDB 3IOQ) ranks as one of the least basic cysteine isoforms from C. candamarcensis, is structurally closer to papain, caricain, chymopapain and mexicain than to the other cysteine proteinases, while its activity is twice the activity of papain towards BAPNA substrate. Two differences, one in the S2 subsite and another in the S3 subsite of CMS1MS2 may contribute to the enhanced activity relative to papain. In addition, the model provides a structural basis for the sensitivity of CMS1MS2 to inhibition by cystatin, not shown by other enzymes of the group, e.g., glycyl endopeptidase and CMS2MS2.

  8. Proteolytic factors in exosomes.

    PubMed

    Shimoda, Masayuki; Khokha, Rama

    2013-05-01

    Exosomes are small microvesicles secreted from the late endosomal compartment of cells. Although an increasing body of evidence indicates that they play a pivotal role in cell-to-cell communication, the biological functions of exosomes are far from fully understood. Recent work has revealed detailed proteomic profiles of exosomes from cell lines and body fluids, which may provide clues to understanding their biological significance and general importance in human diseases. Metalloproteinases include the cell surface-anchored sheddases a disintegrin and metalloproteinases, as well as cell surface-bound and soluble matrix metalloproteinases and these extracellular proteases have been detected in exosomes by proteomic analyses. Exosomes play a key role in the transfer of proteins to other cells and metalloproteinases may provide a novel platform where ectodomain shedding by these membrane proteases alters the makeup of the recipient cell's surface. This review aims to address some of the facets of exosome biology with particular emphasis on the proteolytic factors and we discuss their potential involvement in human diseases, especially tumor biology.

  9. Ca/sup 2 +/-dependent proteolytic activity in crab claw muscle: effects of inhibitors and specificity for myofibrillar proteins

    SciTech Connect

    Mykles, D.L.; Skinner, D.M.

    1983-09-10

    The claw closer muscle of the Bermuda land crab, Gecarcinus lateralis, undergoes a sequential atrophy and restoration during each molting cycle. The role of Ca/sup 2 +/-dependent proteinases in the turn-over of myofibrillar protein in normal anecdysial (intermolt) claw muscle is described. Crab Ca/sup 2 +/-dependent proteinase degrades the myofibrillar proteins actin, myosin heavy and light chains, paramyosin, tropomyosin, and troponin-T and -I. Ca/sup 2 +/-dependent proteinase activity in whole homogenates and 90,000 x g supernatant fractions from muscle homogenates has been characterized with respect to Ca/sup 2 +/ requirement, substrate specificity, and effects of proteinase inhibitors. The enzyme is inhibited by antipain, leupeptin, E-64, and iodoacetamide; it is insensitive to pepstatin A. The specificity of crab Ca/sup 2 +/-dependent proteinase was examined with native myosin with normal ATPase activity as well as with radioiodinated myosin and radioiodinated hemolymph proteins. Hydrolysis of /sup 125/I-myosin occurs in two phases, both Ca/sup 2 +/-dependent: (1) heavy chain (M/sub r/ = 200,000) is cleaved into four large fragments (M/sub r/ = 160,000, 110,000, 73,000, 60,000) and numerous smaller fragments; light chain (M/sub r/ = 18,000) is cleaved to a 15,000-Da fragment; (2) the fragments produced in the first phase are hydrolyzed to acid-soluble material. Although radioiodinated native hemolymph proteins are not susceptible to the Ca/sup 2 +/-dependent proteinase, those denatured by carboxymethylation are degraded. These data suggest that crab Ca/sup 2 +/-dependent proteinase is involved in turnover of myofibrillar protein in normal muscle and muscle undergoing proecdysial atrophy.

  10. Ubiquitin-dependent proteolytic pathway in wheat germ: Isolation of multiple forms of ubiquitin-activating enzyme, E1

    SciTech Connect

    Hatfield, P.M.; Vierstra, R.D. )

    1989-01-24

    Ubiquitin is a highly conserved protein involved in several important regulatory processes through its ATP-dependent, covalent ligation to a variety of eukaryotic target proteins. The authors describe here the characterization of ubiquitin conjugation in wheat germ extracts and the subsequent isolation of enzymes involved in conjugation. With {sup 125}I-ubiquitin as a substrate, wheat germ extracts form conjugates with either endogenous or added proteins. Ubiquitin-activating enzyme (E1) was purified from wheat germ extracts by using a modification of the covalent affinity chromatography procedure of Ciechanover et al. E1 from wheat germ, like that from rabbit reticulocytes, formed thiol ester intermediates with ubiquitin in the presence of ATP. Purified E1 preparations contained three polypeptides of apparent molecular masses of 117, 123, and 126 kDa after NaDodSO{sub 4}-PAGE. Under nondenaturing conditions, these proteins have native molecular masses of {approx}115 kDa, indicating that they exist as monomers. They concluded that all three species were E1 on the basis of their coelution with E1 activity, by immunorecognition by anti-human E1 antibodies, and by the similarity of their peptide maps. Furthermore, antibodies prepared against wheat germ E1's recognized E1 from rabbit reticulocytes. All three wheat germ E1's were detected in crude extracts prepared under conditions that minimized proteolysis, suggesting that the heterogeneity of the purified E1 preparations was not the result of posthomogenization breakdown. The immunological similarity of animal and plant E1's indicates that this conjugation enzyme, like ubiquitin, has been conserved through evolution.

  11. The Unusual Resistance of Avian Defensin AvBD7 to Proteolytic Enzymes Preserves Its Antibacterial Activity

    PubMed Central

    Bailleul, Geoffrey; Kravtzoff, Amanda; Joulin-Giet, Alix; Lecaille, Fabien; Labas, Valérie; Meudal, Hervé; Loth, Karine; Teixeira-Gomes, Ana-Paula; Gilbert, Florence B.; Coquet, Laurent; Jouenne, Thierry; Brömme, Dieter; Schouler, Catherine; Landon, Céline; Lalmanach, Gilles; Lalmanach, Anne-Christine

    2016-01-01

    Defensins are frontline peptides of mucosal immunity in the animal kingdom, including birds. Their resistance to proteolysis and their ensuing ability to maintain antimicrobial potential remains questionable and was therefore investigated. We have shown by bottom-up mass spectrometry analysis of protein extracts that both avian beta-defensins AvBD2 and AvBD7 were ubiquitously distributed along the chicken gut. Cathepsin B was found by immunoblotting in jejunum, ileum, caecum, and caecal tonsils, while cathepsins K, L, and S were merely identified in caecal tonsils. Hydrolysis product of AvBD2 and AvBD7 incubated with a panel of proteases was analysed by RP-HPLC, mass spectrometry and antimicrobial assays. AvBD2 and AvBD7 were resistant to serine proteases and to cathepsins D and H. Conversely cysteine cathepsins B, K, L, and S degraded AvBD2 and abolished its antibacterial activity. Only cathepsin K cleaved AvBD7 and released Ile4-AvBD7, a N-terminal truncated natural peptidoform of AvBD7 that displayed antibacterial activity. Besides the 3-stranded antiparallel beta-sheet typical of beta-defensins, structural analysis of AvBD7 by two-dimensional NMR spectroscopy highlighted the restricted accessibility of the C-terminus embedded by the N-terminal region and gave a formal evidence of a salt bridge (Asp9-Arg12) that could account for proteolysis resistance. The differential susceptibility of avian defensins to proteolysis opens intriguing questions about a distinctive role in the mucosal immunity against pathogen invasion. PMID:27561012

  12. The proteolytically stable peptidomimetic Pam-(Lys-βNSpe)6-NH2 selectively inhibits human neutrophil activation via formyl peptide receptor 2.

    PubMed

    Skovbakke, Sarah Line; Heegaard, Peter M H; Larsen, Camilla J; Franzyk, Henrik; Forsman, Huamei; Dahlgren, Claes

    2015-01-15

    Immunomodulatory host defense peptides (HDPs) are considered to be lead compounds for novel anti-sepsis and anti-inflammatory agents. However, development of drugs based on HDPs has been hampered by problems with toxicity and low bioavailability due to in vivo proteolysis. Here, a subclass of proteolytically stable HDP mimics consisting of lipidated α-peptide/β-peptoid oligomers was investigated for their effect on neutrophil function. The most promising compound, Pam-(Lys-βNSpe)6-NH2, was shown to inhibit formyl peptide receptor 2 (FPR2) agonist-induced neutrophil granule mobilization and release of reactive oxygen species. The potency of Pam-(Lys-βNSpe)6-NH2 was comparable to that of PBP10, the most potent FPR2-selective inhibitor known. The immunomodulatory effects of structural analogs of Pam-(Lys-βNSpe)6-NH2 emphasized the importance of both the lipid and peptidomimetic parts. By using imaging flow cytometry in primary neutrophils and FPR-transfected cell lines, we found that a fluorescently labeled analog of Pam-(Lys-βNSpe)6-NH2 interacted selectively with FPR2. Furthermore, the interaction between Pam-(Lys-βNSpe)6-NH2 and FPR2 was found to prevent binding of the FPR2-specific activating peptide agonist Cy5-WKYMWM, while the binding of an FPR1-selective agonist was not inhibited. To our knowledge, Pam-(Lys-βNSpe)6-NH2 is the first HDP mimic found to inhibit activation of human neutrophils via direct interaction with FPR2. Hence, we consider Pam-(Lys-βNSpe)6-NH2 to be a convenient tool in the further dissection of the role of FPR2 in inflammation and homeostasis as well as for investigation of the importance of neutrophil stimulation in anti-infective therapy involving HDPs. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Synthesis of a novel chitosan-based Ce(IV) complex with proteolytic activity in vitro toward edible biological proteins.

    PubMed

    Li, Yan; Liu, Bingjie; Liu, Zihui; Meng, Xianghong; Wang, Dongfeng

    2016-04-20

    The occurrence of enzymatic activities is attributed to proper spatial organization of functional groups from first principles. A novel chitosan-based Ce(IV) complex (CC[Ce(IV)]), an artificial metalloproteinase, was synthesized by attaching cyclen, Ce(IV), and chlorophyll-Cu(II) to a chitosan-based matrix. The enzymatic hydrolytic efficiency (HE) and the procedure of catalyzing myoglobin (Mb) by CC[Ce(IV)] in vitro were investigated using spectrophotometry, electrophoresis, and liquid chromatography. The results showed that the HE of Mb was up to 60% at 60°C within 24h, displaying a catalytic proficiency. The pseudo-first-order kinetic constant (kobs) for CC[Ce(IV)] treatment within 24h was 3.85×10(-2)h(-1), higher than that for α-chymotrypsin treatment, which was 2.63×10(-2)h(-1). Moreover, the peptide bond derived from Asp-Phe/Phe-Asp in Mb could be specifically cleaved by CC[Ce(IV)], which could simulate the functionality of α-chymotrypsin. This work provides an experimental basis for potential utilization of the chitosan-based Ce(IV) complexes in the food industry. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Inhibitory potential of three zinc chelating agents against the proteolytic, hemorrhagic, and myotoxic activities of Echis carinatus venom.

    PubMed

    Nanjaraj Urs, Ankanahalli N; Yariswamy, Manjunath; Ramakrishnan, Chandrasekaran; Joshi, Vikram; Suvilesh, Kanve Nagaraj; Savitha, Mysore Natarajan; Velmurugan, Devadasan; Vishwanath, Bannikuppe Sannanayak

    2015-01-01

    Viperbites undeniably cause local manifestations such as hemorrhage and myotoxicity involving substantial degradation of extracellular matrix (ECM) at the site of envenomation and lead to progressive tissue damage and necrosis. The principle toxin responsible is attributed to snake venom metalloproteases (SVMPs). Treatment of such progressive tissue damage induced by SVMPs has become a challenging task for researchers and medical practitioners who are in quest of SVMPs inhibitors. In this study, we have evaluated the inhibitory potential of three specific zinc (Zn(2+)) chelating agents; N,N,N',N'-tetrakis (2-pyridylmethyl) ethane-1,2-diamine (TPEN), diethylene triamine pentaacetic acid (DTPA), tetraethyl thiuram disulfide (TTD) on Echis carinatus venom (ECV) induced hemorrhage and myotoxicity. Amongst them, TPEN has high affinity for Zn(2+) and revealed potent inhibition of ECV metalloproteases (ECVMPs) in vitro (IC50: 6.7 μM) compared to DTPA and TTD. The specificity of TPEN towards Zn(2+) was confirmed by spectral and docking studies. Further, TPEN, DTPA, and TTD completely blocked the hemorrhagic and myotoxic activities of ECV in a dose dependent manner upon co-injection; whereas, only TPEN successfully neutralized hemorrhage and myotoxicity following independent injection. Histological examinations revealed that TPEN effectively prevents degradation of dermis and basement membrane surrounding the blood vessels in mouse skin sections. TPEN also prevents muscle necrosis and accumulation of inflammatory cells at the site of ECV injections. In conclusion, a high degree of structural and functional homology between mammalian MMPs and SVMPs suggests that specific Zn(2+) chelators currently in clinical practice could be potent first aid therapeutic agents in snakebite management, particularly for local tissue damage. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Ectopically Expressed Pro-group X Secretory Phospholipase A2 Is Proteolytically Activated in Mouse Adrenal Cells by Furin-like Proprotein Convertases

    PubMed Central

    Layne, Joseph D.; Shridas, Preetha; Webb, Nancy R.

    2015-01-01

    Group X secretory phospholipase A2 (GX sPLA2) hydrolyzes mammalian cell membranes, liberating free fatty acids and lysophospholipids. GX sPLA2 is produced as a pro-enzyme (pro-GX sPLA2) that contains an N-terminal 11-amino acid propeptide ending in a dibasic motif, suggesting cleavage by a furin-like proprotein convertase (PC). Although propeptide cleavage is clearly required for enzymatic activity, the protease(s) responsible for pro-GX sPLA2 activation have not been identified. We previously reported that GX sPLA2 negatively regulates adrenal glucocorticoid production, likely by suppressing liver X receptor-mediated activation of steroidogenic acute regulatory protein expression. In this study, using a FLAG epitope-tagged pro-GX sPLA2 expression construct (FLAG-pro-GX sPLA2), we determined that adrenocorticotropic hormone (ACTH) enhanced FLAG-pro-GX sPLA2 processing and phospholipase activity secreted by Y1 adrenal cells. ACTH increased the expression of furin and PCSK6, but not other members of the PC family, in Y1 cells. Overexpression of furin and PCSK6 in HEK 293 cells significantly enhanced FLAG-pro-GX sPLA2 processing, whereas siRNA-mediated knockdown of both PCs almost completely abolished FLAG-pro-GX sPLA2 processing in Y1 cells. Expression of either furin or PCSK6 enhanced the ability of GX sPLA2 to suppress liver X receptor reporter activity. The PC inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone significantly suppressed FLAG-pro-GX sPLA2 processing and sPLA2 activity in Y1 cells, and it significantly attenuated GX sPLA2-dependent inhibition of steroidogenic acute regulatory protein expression and progesterone production. These findings provide strong evidence that pro-GX sPLA2 is a substrate for furin and PCSK6 proteolytic processing and define a novel mechanism for regulating corticosteroid production in adrenal cells. PMID:25623068

  16. Proteolytic activation and function of the cytokine Spätzle in innate immune response of a lepidopteran insect, Manduca sexta

    PubMed Central

    An, Chunju; Jiang, Haobo; Kanost, Michael R.

    2009-01-01

    The innate immune response of insects includes induced expression of genes encoding a variety of antimicrobial peptides. The signaling pathways that stimulate this gene expression have been well characterized by genetic analysis in Drosophila melanogaster, but are not well understood in most other insect species. One such pathway involves proteolytic activation of a cytokine called Spätzle, which functions in dorsal-ventral patterning in early embryonic development and in the antimicrobial immune response in larvae and adults. We have investigated the function of Spätzle in a lepidopteran insect, Manduca sexta, in which hemolymph proteinases activated during immune responses have been characterized biochemically. Two cDNA isoforms for M. sexta Spätzle-1 differ due to alternative splicing, resulting in a 10 amino acid residue insertion in the pro-region of proSpätzle-1B that is not present in proSpätzle-1A. The proSpätzle-1A cDNA encodes a 32.7 kDa polypeptide that is 23% and 44% identical to D. melanogaster and Bombyx mori Spätzle-1, respectively. Recombinant proSpätzle-1A was a disulfide-linked homodimer. M. sexta hemolymph proteinase 8 (HP8) cleaved proSpätzle-1A to release Spätzle-C108, a dimer of the carboxyl-terminal 108-residue cystine-knot domain. Injection of Spätzle-C108, but not proSpätzle-1A, into larvae stimulated expression of several antimicrobial peptides and proteins, including attacin-1, cecropin-6, moricin, lysozyme, and the immunoglobulin domain protein hemolin, but did not significantly affect expression of two bacteria-inducible pattern recognition proteins, immulectin-2 and β-1,3-glucan recognition protein-2. Results from this paper and other recent studies support a model for a pathway in which the clip-domain proteinase proHP6 becomes activated in plasma upon exposure to Gram-negative or Gram-positive bacteria or to β-1,3-glucan. HP6 then activates proHP8, which in turn activates Spätzle-1. The resulting Spätzle-C108 dimer is

  17. Myostatin induces cachexia by activating the ubiquitin proteolytic system through an NF-kappaB-independent, FoxO1-dependent mechanism.

    PubMed

    McFarlane, Craig; Plummer, Erin; Thomas, Mark; Hennebry, Alex; Ashby, Murray; Ling, Nicholas; Smith, Heather; Sharma, Mridula; Kambadur, Ravi

    2006-11-01

    Myostatin, a transforming growth factor-beta (TGF-beta) super-family member, has been well characterized as a negative regulator of muscle growth and development. Myostatin has been implicated in several forms of muscle wasting including the severe cachexia observed as a result of conditions such as AIDS and liver cirrhosis. Here we show that Myostatin induces cachexia by a mechanism independent of NF-kappaB. Myostatin treatment resulted in a reduction in both myotube number and size in vitro, as well as a loss in body mass in vivo. Furthermore, the expression of the myogenic genes myoD and pax3 was reduced, while NF-kappaB (the p65 subunit) localization and expression remained unchanged. In addition, promoter analysis has confirmed Myostatin inhibition of myoD and pax3. An increase in the expression of genes involved in ubiquitin-mediated proteolysis is observed during many forms of muscle wasting. Hence we analyzed the effect of Myostatin treatment on proteolytic gene expression. The ubiquitin associated genes atrogin-1, MuRF-1, and E214k were upregulated following Myostatin treatment. We analyzed how Myostatin may be signaling to induce cachexia. Myostatin signaling reversed the IGF-1/PI3K/AKT hypertrophy pathway by inhibiting AKT phosphorylation thereby increasing the levels of active FoxO1, allowing for increased expression of atrophy-related genes. Therefore, our results suggest that Myostatin induces cachexia through an NF-kappaB-independent mechanism. Furthermore, increased Myostatin levels appear to antagonize hypertrophy signaling through regulation of the AKT-FoxO1 pathway.

  18. Does transgenic Cry1Ac + CpTI cotton pollen affect hypopharyngeal gland development and midgut proteolytic enzyme activity in the honey bee Apis mellifera L. (Hymenoptera, Apidae)?

    PubMed

    Han, Peng; Niu, Chang-Ying; Biondi, Antonio; Desneux, Nicolas

    2012-11-01

    The transgenic Cry1Ac (Bt toxin) + CpTI (Cowpea Trypsin Inhibitor) cotton cultivar CCRI41 is increasingly used in China and potential side effects on the honey bee Apis mellifera L. have been documented recently. Two studies have assessed potential lethal and sublethal effects in young bees fed with CCRI41 cotton pollen but no effect was observed on learning capacities, although lower feeding activity in exposed honey bees was noted (antifeedant effect). The present study aimed at providing further insights into potential side effects of CCRI41 cotton on honey bees. Emerging honey bees were exposed to different pollen diets using no-choice feeding protocols (chronic exposure) in controlled laboratory conditions and we aimed at documenting potential mechanisms underneath the CCRI41 antifeedant effect previously reported. Activity of midgut proteolytic enzyme of young adult honey bees fed on CCRI41 cotton pollen were not significantly affected, i.e. previously observed antifeedant effect was not linked to disturbed activity of the proteolytic enzymes in bees' midgut. Hypopharyngeal gland development was assessed by quantifying total extractable proteins from the glands. Results suggested that CCRI41 cotton pollen carries no risk to hypopharyngeal gland development of young adult honey bees. In the two bioassays, honey bees exposed to 1 % soybean trypsin inhibitor were used as positive controls for both midgut proteolytic enzymes and hypopharyngeal gland proteins quantification, and bees exposed to 48 ppb (part per billion) (i.e. 48 ng g(-1)) imidacloprid were used as controls for exposure to a sublethal concentration of toxic product. The results show that the previously reported antifeedant effect of CCRI41 cotton pollen on honey bees is not linked to effects on their midgut proteolytic enzymes or on the development of their hypopharyngeal glands. The results of the study are discussed in the framework of risk assessment of transgenic crops on honey bees.

  19. Regulation of STAT signalling by proteolytic processing.

    PubMed

    Hendry, Lisa; John, Susan

    2004-12-01

    Interaction of cytokines with their cognate receptors leads to the activation of latent transcription factors, the signal transducer and activator of transcription (STAT) proteins. Numerous studies have identified the critical roles played by STAT proteins in regulating cell proliferation, differentiation and survival. Consequently, the activity of STAT proteins is negatively regulated by a variety of different mechanisms, which include alternative splicing, covalent modifications, protein-protein interactions with negative regulatory proteins and proteolytic processing by proteases. Cleavage of STAT proteins by proteases results in the generation of C-terminally truncated proteins, called STATgamma, which lack the transactivation domain and behave as functional dominant-negative proteins. Currently, STATgamma isoforms have been identified for Stat3, Stat5a, Stat5b and Stat6 in different cellular contexts and biological processes. Evidence is mounting for the role of as yet unidentified serine proteases in the proteolytic processing of STAT proteins, although at least one cysteine protease, calpain is also known to cleave these STATs in platelets and mast cells. Recently, studies of acute myeloid leukaemia and cutaneous T cell lymphoma patients have revealed important roles for the aberrant expression of Stat3gamma and Stat5gamma proteins in the pathology of these diseases. Together, these findings indicate that proteolytic processing is an important mechanism in the regulation of STAT protein biological activity and provides a fertile area for future studies.

  20. Development of a novel fluorogenic proteolytic beacon for in vivo detection and imaging of tumour-associated matrix metalloproteinase-7 activity.

    PubMed Central

    McIntyre, J Oliver; Fingleton, Barbara; Wells, K Sam; Piston, David W; Lynch, Conor C; Gautam, Shiva; Matrisian, Lynn M

    2004-01-01

    The present study describes the in vivo detection and imaging of tumour-associated MMP-7 (matrix metalloproteinase-7 or matrilysin) activity using a novel polymer-based fluorogenic substrate PB-M7VIS, which serves as a selective 'proteolytic beacon' (PB) for this metalloproteinase. PB-M7VIS is built on a PAMAM (polyamido amino) dendrimer core of 14.2 kDa, covalently coupled with an Fl (fluorescein)-labelled peptide Fl(AHX)RPLALWRS(AHX)C (where AHX stands for aminohexanoic acid) and with TMR (tetramethylrhodamine). PB-M7VIS is efficiently and selectively cleaved by MMP-7 with a k (cat)/ K (m) value of 1.9x10(5) M(-1).s(-1) as measured by the rate of increase in Fl fluorescence (up to 17-fold for the cleavage of an optimized PB-M7VIS) with minimal change in the TMR fluorescence. The K (m) value for PB-M7VIS is approx. 0.5 microM, which is approx. two orders of magnitude lower when compared with that for an analogous soluble peptide, indicating efficient interaction of MMP-7 with the synthetic polymeric substrate. With MMP-2 or -3, the k (cat)/ K (m) value for PB-M7VIS is approx. 56- or 13-fold lower respectively, when compared with MMP-7. In PB-M7VIS, Fl(AHX)RPLALWRS(AHX)C is a selective optical sensor of MMP-7 activity and TMR serves to detect both the uncleaved and cleaved reagents. Each of these can be visualized as subcutaneous fluorescent phantoms in a mouse and optically discriminated based on the ratio of green/red (Fl/TMR) fluorescence. The in vivo specificity of PB-M7VIS was tested in a mouse xenograft model. Intravenous administration of PB-M7VIS gave significantly enhanced Fl fluorescence from MMP-7-positive tumours, but not from control tumours ( P <0.0001), both originally derived from SW480 human colon cancer cells. Prior systemic treatment of the tumour-bearing mice with an MMP inhibitor BB-94 ([4-( N -hydroxyamino)-2 R -isobutyl-3 S -(thienylthiomethyl)-succinyl]-L-phenylalanine- N -methylamide), markedly decreased the Fl fluorescence over the MMP-7

  1. Dual Function of Novel Pollen Coat (Surface) Proteins: IgE-binding Capacity and Proteolytic Activity Disrupting the Airway Epithelial Barrier

    PubMed Central

    Bashir, Mohamed Elfatih H.; Ward, Jason M.; Cummings, Matthew; Karrar, Eltayeb E.; Root, Michael; Mohamed, Abu Bekr A.; Naclerio, Robert M.; Preuss, Daphne

    2013-01-01

    Background The pollen coat is the first structure of the pollen to encounter the mucosal immune system upon inhalation. Prior characterizations of pollen allergens have focused on water-soluble, cytoplasmic proteins, but have overlooked much of the extracellular pollen coat. Due to washing with organic solvents when prepared, these pollen coat proteins are typically absent from commercial standardized allergenic extracts (i.e., “de-fatted”), and, as a result, their involvement in allergy has not been explored. Methodology/Principal Findings Using a unique approach to search for pollen allergenic proteins residing in the pollen coat, we employed transmission electron microscopy (TEM) to assess the impact of organic solvents on the structural integrity of the pollen coat. TEM results indicated that de-fatting of Cynodon dactylon (Bermuda grass) pollen (BGP) by use of organic solvents altered the structural integrity of the pollen coat. The novel IgE-binding proteins of the BGP coat include a cysteine protease (CP) and endoxylanase (EXY). The full-length cDNA that encodes the novel IgE-reactive CP was cloned from floral RNA. The EXY and CP were purified to homogeneity and tested for IgE reactivity. The CP from the BGP coat increased the permeability of human airway epithelial cells, caused a clear concentration-dependent detachment of cells, and damaged their barrier integrity. Conclusions/Significance Using an immunoproteomics approach, novel allergenic proteins of the BGP coat were identified. These proteins represent a class of novel dual-function proteins residing on the coat of the pollen grain that have IgE-binding capacity and proteolytic activity, which disrupts the integrity of the airway epithelial barrier. The identification of pollen coat allergens might explain the IgE-negative response to available skin-prick-testing proteins in patients who have positive symptoms. Further study of the role of these pollen coat proteins in allergic responses is

  2. Proteolytic Pathways Induced by Herbicides That Inhibit Amino Acid Biosynthesis

    PubMed Central

    Zulet, Amaia; Gil-Monreal, Miriam; Villamor, Joji Grace; Zabalza, Ana; van der Hoorn, Renier A. L.; Royuela, Mercedes

    2013-01-01

    Background The herbicides glyphosate (Gly) and imazamox (Imx) inhibit the biosynthesis of aromatic and branched-chain amino acids, respectively. Although these herbicides inhibit different pathways, they have been reported to show several common physiological effects in their modes of action, such as increasing free amino acid contents and decreasing soluble protein contents. To investigate proteolytic activities upon treatment with Gly and Imx, pea plants grown in hydroponic culture were treated with Imx or Gly, and the proteolytic profile of the roots was evaluated through fluorogenic kinetic assays and activity-based protein profiling. Results Several common changes in proteolytic activity were detected following Gly and Imx treatment. Both herbicides induced the ubiquitin-26 S proteasome system and papain-like cysteine proteases. In contrast, the activities of vacuolar processing enzymes, cysteine proteases and metacaspase 9 were reduced following treatment with both herbicides. Moreover, the activities of several putative serine protease were similarly increased or decreased following treatment with both herbicides. In contrast, an increase in YVADase activity was observed under Imx treatment versus a decrease under Gly treatment. Conclusion These results suggest that several proteolytic pathways are responsible for protein degradation upon herbicide treatment, although the specific role of each proteolytic activity remains to be determined. PMID:24040092

  3. Lactobacillus helveticus: the proteolytic system

    PubMed Central

    Griffiths, M. W.; Tellez, A. M.

    2012-01-01

    Lactobacillus helveticus is one of the species of lactic acid bacteria (LAB) most commonly used in the production of fermented milk beverages and some types of hard cheese. The versatile nature of this bacterium is based on its highly efficient proteolytic system consisting of cell-envelope proteinases (CEPs), transport system and intracellular peptidases. Besides use of L. helveticus in cheese processing, the production of fermented milk preparations with health promoting properties has become an important industrial application. Studies have shown that fermented dairy products are able to decrease blood pressure, stimulate the immune system, promote calcium absorption, and exert an anti-virulent effect against pathogens. These beneficial effects are produced by a variety of peptides released during the hydrolysis of milk proteins by the proteolytic system of L. helveticus, which provides the bacterium with its nutritional requirements for growth. In recent years, studies have focused on understanding the factors that affect the kinetics of milk protein hydrolysis by specific strains and have concentrated on the effect of pH, temperature, growth phase, and matrix composition on the bacterial enzymatic system. This review focuses on the role of the proteolytic system of L. helveticus in the production of bioactive compounds formed during fermentation of dairy products. Taking advantage of the powerful proteolytic system of this bacterium opens up future opportunities to search for novel food-derived compounds with potential health promoting properties. PMID:23467265

  4. [Some properties of proteolytic enzymes from Tribolium confusum].

    PubMed

    Balaian, V M; Levitskiĭ, A P

    1982-01-01

    The digestive tract of Tribolium confusum Duv. larvae was studied for proteolytic enzymes properties. The pH optima are determined for the enzymes effect on various substrates. Proteases were partially purified by gel chromatography on Sephadex G = 100 and investigated for thyol compound influence on their activity. The activity of the enzymes is shown to increase considerably with addition of cystein, glutathione, 2-mercaptoethanol. dithiotreitol and EDTA. Dithiotreitol produces the strongest restoring effect and in concentration of 10(-6) M it activates the enzyme almost twice. Storage for 48 h at 4 degrees C induced a 2.5-fold decrease in the proteolytic enzyme activity; SH-groups in the catalytic action of enzymic solutions is shown. The maximum proteolytic activity is found in extracts from 14-day insects.

  5. Single Cell Proteolytic Assays to Investigate Cancer Clonal Heterogeneity and Cell Dynamics Using an Efficient Cell Loading Scheme

    NASA Astrophysics Data System (ADS)

    Chen, Yu-Chih; Cheng, Yu-Heng; Ingram, Patrick; Yoon, Euisik

    2016-06-01

    Proteolytic degradation of the extracellular matrix (ECM) is critical in cancer invasion, and recent work suggests that heterogeneous cancer populations cooperate in this process. Despite the importance of cell heterogeneity, conventional proteolytic assays measure average activity, requiring thousands of cells and providing limited information about heterogeneity and dynamics. Here, we developed a microfluidic platform that provides high-efficiency cell loading and simple valveless isolation, so the proteolytic activity of a small sample (10–100 cells) can be easily characterized. Combined with a single cell derived (clonal) sphere formation platform, we have successfully demonstrated the importance of microenvironmental cues for proteolytic activity and also investigated the difference between clones. Furthermore, the platform allows monitoring single cells at multiple time points, unveiling different cancer cell line dynamics in proteolytic activity. The presented tool facilitates single cell proteolytic analysis using small samples, and our findings illuminate the heterogeneous and dynamic nature of proteolytic activity.

  6. Single Cell Proteolytic Assays to Investigate Cancer Clonal Heterogeneity and Cell Dynamics Using an Efficient Cell Loading Scheme

    PubMed Central

    Chen, Yu-Chih; Cheng, Yu-Heng; Ingram, Patrick; Yoon, Euisik

    2016-01-01

    Proteolytic degradation of the extracellular matrix (ECM) is critical in cancer invasion, and recent work suggests that heterogeneous cancer populations cooperate in this process. Despite the importance of cell heterogeneity, conventional proteolytic assays measure average activity, requiring thousands of cells and providing limited information about heterogeneity and dynamics. Here, we developed a microfluidic platform that provides high-efficiency cell loading and simple valveless isolation, so the proteolytic activity of a small sample (10–100 cells) can be easily characterized. Combined with a single cell derived (clonal) sphere formation platform, we have successfully demonstrated the importance of microenvironmental cues for proteolytic activity and also investigated the difference between clones. Furthermore, the platform allows monitoring single cells at multiple time points, unveiling different cancer cell line dynamics in proteolytic activity. The presented tool facilitates single cell proteolytic analysis using small samples, and our findings illuminate the heterogeneous and dynamic nature of proteolytic activity. PMID:27283981

  7. Allyl isothiocyanate suppresses the proteolytic activation of sterol regulatory element-binding proteins and de novo fatty acid and cholesterol synthesis.

    PubMed

    Miyata, Shingo; Inoue, Jun; Shimizu, Makoto; Sato, Ryuichiro

    2016-05-01

    Sterol regulatory element-binding proteins (SREBPs) are a family of transcription factors that regulate lipid homeostasis by controlling the expression of genes involved in fatty acid and cholesterol synthesis. In this study, we used a stable cell line that expresses a luciferase reporter gene driven by an SRE-containing fatty acid synthase promoter to identify allyl isothiocyanate (AITC), one of the major isothiocyanates in cruciferous vegetables, as a novel SREBP inactivator. We found that AITC downregulated the proteolytic processing of SREBPs and the expression of their target genes in human hepatoma Huh-7 cells. Furthermore, AITC reduced the de novo synthesis of both fatty acids and cholesterol. Our results indicate a novel physiological function of AITC in lipid metabolism regulation.

  8. Analysis of proteolytic processes and enzymatic activities in the generation of huntingtin n-terminal fragments in an HEK293 cell model.

    PubMed

    Tebbenkamp, Andrew T N; Crosby, Keith W; Siemienski, Zoe B; Brown, Hilda H; Golde, Todd E; Borchelt, David R

    2012-01-01

    N-terminal fragments of mutant huntingtin (htt) that terminate between residues 90-115, termed cleavage product A or 1 (cp-A/1), form intracellular and intranuclear inclusion bodies in the brains of patients with Huntington's disease (HD). These fragments appear to be proteolytic products of the full-length protein. Here, we use an HEK293 cell culture model to investigate huntingtin proteolytic processing; previous studies of these cells have demonstrated cleavage of htt to cp-A/1 like htt fragments. Recombinant N-terminal htt fragments, terminating at residue 171 (also referred to as cp-B/2 like), were efficiently cleaved to produce cp-A/1 whereas fragments representing endogenous caspase, calpain, and metalloproteinase cleavage products, terminating between residues 400-600, were inefficiently cleaved. Using cysteine-labeling techniques and antibody binding mapping, we localized the C-terminus of the cp-A/1 fragments produced by HEK293 cells to sequences minimally limited by cysteine 105 and an antibody epitope composed of residues 115-124. A combination of genetic and pharmacologic approaches to inhibit potential proteases, including γ-secretase and calpain, proved ineffective in preventing production of cp-A/1. Our findings indicate that HEK293 cells express a protease that is capable of efficiently cleaving cp-B/2 like fragments of htt with normal or expanded glutamine repeats. For reasons that remain unclear, this protease cleaves longer htt fragments, with normal or expanded glutamine expansions, much less efficiently. The protease in HEK293 cells that is capable of generating a cp-A/1 like htt fragment may be a novel protease with a high preference for a cp-B/2-like htt fragment as substrate.

  9. Essentials of Proteolytic Machineries in Chloroplasts.

    PubMed

    Nishimura, Kenji; Kato, Yusuke; Sakamoto, Wataru

    2017-01-09

    Plastids are unique organelles that can alter their structure and function in response to environmental and developmental stimuli. Chloroplasts are one type of plastid and are the sites for various metabolic processes, including photosynthesis. For optimal photosynthetic activity, the chloroplast proteome must be properly shaped and maintained through regulated proteolysis and protein quality control mechanisms. Enzymatic functions and activities are conferred by protein maturation processes involving consecutive proteolytic reactions. Protein abundances are optimized by the balanced protein synthesis and degradation, which is depending on the metabolic status. Malfunctioning proteins are promptly degraded. Twenty chloroplast proteolytic machineries have been characterized to date. Specifically, processing peptidases and energy-driven processive proteases are the major players in chloroplast proteome biogenesis, remodeling, and maintenance. Recently identified putative proteases are potential regulators of photosynthetic functions. Here we provide an updated, comprehensive overview of chloroplast protein degradation machineries and discuss their importance for photosynthesis. Wherever possible, we also provide structural insights into chloroplast proteases that implement regulated proteolysis of substrate proteins/peptides. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

  10. recA mutations that reduce the constitutive coprotease activity of the RecA1202(Prtc) protein: possible involvement of interfilament association in proteolytic and recombination activities.

    PubMed Central

    Liu, S K; Eisen, J A; Hanawalt, P C; Tessman, I

    1993-01-01

    Twenty-eight recA mutants, isolated after spontaneous mutagenesis generated by the combined action of RecA1202(Prtc) and UmuDC proteins, were characterized and sequenced. The mutations are intragenic suppressors of the recA1202 allele and were detected by the reduced coprotease activity of the gene product. Twenty distinct mutation sites were found, among which two mutations, recA1620 (V-275-->D) and recA1631 (I-284-->N), were mapped in the C-terminal portion of the interfilament contact region (IFCR) in the RecA crystal. An interaction of this region with the part of the IFCR in which the recA1202 mutation (Q-184-->K) is mapped could occur only intermolecularly. Thus, altered IFCR and the likely resulting change in interfilament association appear to be important aspects of the formation of a constitutively active RecA coprotease. This observation is consistent with the filament-bundle theory (R. M. Story, I. T. Weber, and T. A. Steitz, Nature (London) 335:318-325, 1992). Furthermore, we found that among the 20 suppressor mutations, 3 missense mutations that lead to recombination-defective (Rec-) phenotypes also mapped in the IFCR, suggesting that the IFCR, with its putative function in interfilament association, is required for the recombinase activity of RecA. We propose that RecA-DNA complexes may form bundles analogous to the RecA bundles (lacking DNA) described by Story et al. and that these RecA-DNA bundles play a role in homologous recombination. Images PMID:8407828

  11. Proteolytic Inhibition of Salmonella enterica Serovar Typhimurium-Induced Activation of the Mitogen-Activated Protein Kinases ERK and JNK in Cultured Human Intestinal Cells

    PubMed Central

    Mynott, Tracey L.; Crossett, Ben; Prathalingam, S. Radhika

    2002-01-01

    Bromelain, a mixture of cysteine proteases from pineapple stems, blocks signaling by the mitogen-activated protein (MAP) kinases extracellular regulated kinase 1 (ERK-1) and ERK-2, inhibits inflammation, and protects against enterotoxigenic Escherichia coli infection. In this study, we examined the effect of bromelain on Salmonella enterica serovar Typhimurium infection, since an important feature of its pathogenesis is its ability to induce activation of ERK-1 and ERK-2, which leads to internalization of bacteria and induction of inflammatory responses. Our results show that bromelain dose dependently blocks serovar Typhimurium-induced ERK-1, ERK-2, and c-Jun NH2-terminal kinase (JNK) activation in Caco-2 cells. Bromelain also blocked signaling induced by carbachol and anisomycin, pharmacological MAP kinase agonists. Despite bromelain inhibition of serovar Typhimurium-induced MAP kinase signaling, it did not prevent subsequent invasion of the Caco-2 cells by serovar Typhimurium or alter serovar Typhimurium -induced decreases in resistance across Caco-2 monolayers. Surprisingly, bromelain also did not block serovar Typhimurium-induced interleukin-8 (IL-8) secretion but synergized with serovar Typhimurium to enhance IL-8 production. We also found that serovar Typhimurium does not induce ERK phosphorylation in Caco-2 cells in the absence of serum but that serovar Typhimurium-induced invasion and decreases in monolayer resistance are unaffected. Collectively, these data indicate that serovar Typhimurium-induced invasion of Caco-2 cells, changes in the resistance of epithelial cell monolayers, and IL-8 production can occur independently of the ERK and JNK signaling pathways. Data also confirm that bromelain is a novel inhibitor of MAP kinase signaling pathways and suggest a novel role for proteases as inhibitors of signal transduction pathways in intestinal epithelial cells. PMID:11748167

  12. Proteolytic inhibition of Salmonella enterica serovar typhimurium-induced activation of the mitogen-activated protein kinases ERK and JNK in cultured human intestinal cells.

    PubMed

    Mynott, Tracey L; Crossett, Ben; Prathalingam, S Radhika

    2002-01-01

    Bromelain, a mixture of cysteine proteases from pineapple stems, blocks signaling by the mitogen-activated protein (MAP) kinases extracellular regulated kinase 1 (ERK-1) and ERK-2, inhibits inflammation, and protects against enterotoxigenic Escherichia coli infection. In this study, we examined the effect of bromelain on Salmonella enterica serovar Typhimurium infection, since an important feature of its pathogenesis is its ability to induce activation of ERK-1 and ERK-2, which leads to internalization of bacteria and induction of inflammatory responses. Our results show that bromelain dose dependently blocks serovar Typhimurium-induced ERK-1, ERK-2, and c-Jun NH(2)-terminal kinase (JNK) activation in Caco-2 cells. Bromelain also blocked signaling induced by carbachol and anisomycin, pharmacological MAP kinase agonists. Despite bromelain inhibition of serovar Typhimurium-induced MAP kinase signaling, it did not prevent subsequent invasion of the Caco-2 cells by serovar Typhimurium or alter serovar Typhimurium -induced decreases in resistance across Caco-2 monolayers. Surprisingly, bromelain also did not block serovar Typhimurium-induced interleukin-8 (IL-8) secretion but synergized with serovar Typhimurium to enhance IL-8 production. We also found that serovar Typhimurium does not induce ERK phosphorylation in Caco-2 cells in the absence of serum but that serovar Typhimurium-induced invasion and decreases in monolayer resistance are unaffected. Collectively, these data indicate that serovar Typhimurium-induced invasion of Caco-2 cells, changes in the resistance of epithelial cell monolayers, and IL-8 production can occur independently of the ERK and JNK signaling pathways. Data also confirm that bromelain is a novel inhibitor of MAP kinase signaling pathways and suggest a novel role for proteases as inhibitors of signal transduction pathways in intestinal epithelial cells.

  13. Fas-Induced Apoptosis Increases Hepatocyte Tissue Factor Procoagulant Activity In Vitro and In Vivo

    PubMed Central

    Lopez, Michelle; Kopec, Anna K.; Joshi, Nikita; Geddings, Julia E.; Cline, Holly; Towery, Keara L.; Rockwell, Cheryl E.; Mackman, Nigel; Luyendyk, James P.

    2014-01-01

    Hepatocyte (HPC) apoptosis occurs in association with hepatotoxic responses and chronic liver disease, and is coupled to activation of the blood coagulation cascade. HPCs have been shown to express tissue factor (TF), the primary activator of blood coagulation, in a form that lacks procoagulant activity. In this study, we determined the effect of inducing HPC apoptosis on the procoagulant activity of TF. Treatment of primary mouse HPCs with the Fas death receptor agonist (anti-CD95 antibody, Jo2) triggered apoptosis as shown by cleavage of caspase-3, increased caspase-3 proteolytic activity, and cell surface exposure of phosphatidylserine (PS). Jo2-induced apoptosis significantly increased TF-dependent factor Xa generation by HPCs. Moreover, Jo2 treatment was associated with increased levels of microparticle-associated TF procoagulant activity in the culture medium. Pretreatment with a caspase-3 inhibitor significantly reduced Jo2-induced HPC TF activity and prevented the increase in microparticle-associated TF procoagulant activity. Application of the high-affinity PS-binding protein lactadherin inhibited TF-dependent factor Xa generation by Jo2-treated HPCs and dramatically reduced microparticle-associated TF procoagulant activity. Treatment of wild-type mice with a sublethal dose of Jo2 was associated with a robust increase in the activation of coagulation as measured by plasma thrombin-antithrombin (TAT) levels; whereas mice with liver-specific TF deficiency had significantly lower TAT levels. Overall, the results indicate that Fas-initiated, caspase-3-dependent HPC apoptosis increases TF procoagulant activity through a mechanism involving PS externalization. This suggests that activation of liver TF likely contributes to the procoagulant state associated with HPC apoptosis in liver toxicity and disease. PMID:25015658

  14. Development of a rapid, one-step screening method for the isolation of presumptive proteolytic enterococci.

    PubMed

    Graham, Ken; Rea, Rosemary; Simpson, Paul; Stack, Helena

    2017-01-01

    Enterococci show higher proteolytic activities than other lactic acid bacteria and thus have received considerable attention in scientific literature in recent years. Proteolytic enzymes of enterococci have warranted the use of some species as starter, adjuncts or protective cultures and as probiotics, while in some strains they have also been linked with virulence. Consequently, the isolation and identification of proteolytic enterococci is becoming of increasing interest and importance. However, current screening methods for proteolytic enterococci can be time consuming, requiring a two-step procedure which may take up to 96h. This study describes a method, utilising Kanamycin Skim Milk Aesculin Azide (KSMEA) agar, for the isolation of proteolytic enterococci in one-step, thereby significantly reducing screening time. KSMEA combines the selective properties of Kanamycin Aesculin Azide Agar (KAA) with skim milk powder for the detection of proteolytic enterococci. Enterococci produced colonies with a black halo on KSMEA which were accompanied by a zone of clearing in the media when enterococci were proteolytic. KSMEA medium retained the selectivity of KAA, while proteolytic enterococci were easily distinguished from non-proteolytic enterococci when two known strains were propagated on KSMEA. KSMEA also proved effective at isolating and detecting enterococci in raw milk, faeces and soil. Isolates recovered from the screen were confirmed as enterococci using genus-specific primers. Proteolytic enterococci were present in the raw milk sample only and were easily distinguishable from non-proteolytic enterococci and other microorganisms. Therefore, KSMEA provides a rapid, one-step screening method for the isolation of presumptive proteolytic enterococci.

  15. Near-infrared triple-helical peptide with quenched fluorophores for optical imaging of MMP-2 and MMP-9 proteolytic activity in vivo

    PubMed Central

    Fields, Gregg B.; Edwards, W. Barry

    2014-01-01

    The gelatinase members of the MMP family have consistently been associated with tumor invasiveness, which make them an attractive target for molecular imaging. We report new activatable proteolytic optical imaging agents that consist of triple-helical peptide (THP) conjugates, with high specificity to the gelatinases, bearing quenched cypate dyes. With quenching efficiencies up to 51%, the amplified fluorescence signal upon cypate3-THP hydrolysis by the gelatinases (kcat/KM values of 6.4 × 103 M−1 s−1 to 9.1 × 103 M−1 s−1 for MMP-2 and MMP-9, respectively) in mice bearing human fibrosarcoma xenografted tumors was monitored with fluorescence molecular tomography. There was significant fluorescence enhancement within the tumor and this enhancement was reduced by treatment with pan-MMP inhibitor, Ilomastat. These data, combined with the gelatinase substrate specificity observed in vitro, indicated the observed fluorescence at the site of the tumor was due to gelatinase mediated hydrolysis of cypate3-THP. PMID:25047578

  16. Effects of manganese on tyrosine hydroxylase (TH) activity and TH-phosphorylation in a dopaminergic neural cell line

    SciTech Connect

    Zhang Danhui; Kanthasamy, Arthi; Anantharam, Vellareddy; Kanthasamy, Anumantha

    2011-07-15

    Manganese (Mn) exposure causes manganism, a neurological disorder similar to Parkinson's disease. However, the cellular mechanism by which Mn impairs the dopaminergic neurotransmitter system remains unclear. We previously demonstrated that caspase-3-dependent proteolytic activation of protein kinase C delta (PKC{delta}) plays a key role in Mn-induced apoptotic cell death in dopaminergic neurons. Recently, we showed that PKC{delta} negatively regulates tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, by enhancing protein phosphatase-2A activity in dopaminergic neurons. Here, we report that Mn exposure can affect the enzymatic activity of TH, the rate-limiting enzyme in dopamine synthesis, by activating PKC{delta}-PP2A signaling pathway in a dopaminergic cell model. Low dose Mn (3-10 {mu}M) exposure to differentiated mesencephalic dopaminergic neuronal cells for 3 h induced a significant increase in TH activity and phosphorylation of TH-Ser40. The PKC{delta} specific inhibitor rottlerin did not prevent Mn-induced TH activity or TH-Ser40 phosphorylation. On the contrary, chronic exposure to 0.1-1 {mu}M Mn for 24 h induced a dose-dependent decrease in TH activity. Interestingly, chronic Mn treatment significantly increased PKC{delta} kinase activity and protein phosphatase 2A (PP2A) enzyme activity. Treatment with the PKC{delta} inhibitor rottlerin almost completely prevented chronic Mn-induced reduction in TH activity, as well as increased PP2A activity. Neither acute nor chronic Mn exposures induced any cytotoxic cell death or altered TH protein levels. Collectively, these results demonstrate that low dose Mn exposure impairs TH activity in dopaminergic cells through activation of PKC{delta} and PP2A activity.

  17. Isolation and removal of proteolytic enzymes with magnetic cross-linked erythrocytes

    NASA Astrophysics Data System (ADS)

    Šafařík, Ivo; Šafaříková, Mirka

    2001-01-01

    New magnetic adsorbents for batch isolation and removal of various proteolytic enzymes were prepared by glutaraldehyde cross-linking of bovine, porcine and human erythrocytes in the presence of fine magnetic particles. Trypsin, chymotrypsin, alkaline bacterial protease and proteases present in various commercial enzyme preparations were efficiently adsorbed on these adsorbents; on the contrary, proteins without proteolytic activity were not adsorbed.

  18. Role of Proteolytic Enzymes in the Interaction of Phytopathogenic Microorganisms with Plants.

    PubMed

    Valueva, T A; Zaichik, B Ts; Kudryavtseva, N N

    2016-12-01

    Various forms of participation of proteolytic enzymes in pathogenesis and defense in plants are reviewed. Along with extracellular proteinases, phytopathogenic microorganisms produce specific effectors having proteolytic activity and capable of acting on proteins inside plant cells. In turn, for defense against pathogens, plants use both extracellular and intracellular proteinases.

  19. Isolation and identification of thermophilic and mesophylic proteolytic bacteria from shrimp paste "Terasi"

    NASA Astrophysics Data System (ADS)

    Murwani, R.; Supriyadi, Subagio, Trianto, A.; Ambariyanto

    2015-12-01

    Terasi is a traditional product generally made of fermented shrimp. There were many studies regarding lactic acid bacteria of terasi but none regarding proteolitic bacteria. This study was conducted to isolate and identify the thermophilic and mesophylic proteolytic bacteria from terasi. In addition, the effect of different salt concentrations on the growth of the isolated proteolytic bacteria with the greatest proteolytic activity was also studied. Terasi samples were obtained from the Northern coast region of Java island i.e. Jepara, Demak and Batang. The study obtained 34 proteolytic isolates. Four isolates were identified as Sulfidobacillus, three isolates as Vibrio / Alkaligenes / Aeromonas, two isolates as Pseudomonas, 21 isolates as Bacillus, three isolates as Kurthia/ Caryophanon and one isolates as Amphibacillus. The growth of proteolytic bacteria was affected by salt concentration. The largest growth was found at 0 ppm salt concentrations and growth was declined as salt concentration increased. Maximum growth at each salt concentration tested was found at 8 hours incubation.

  20. Distribution and identification of proteolytic Bacillus spp. in paddy field soil under rice cultivation.

    PubMed

    Watanabe, K; Hayano, K

    1993-07-01

    Proteolytic bacteria in paddy field soils under rice cultivation were characterized and enumerated using azocoll agar plates. Bacillus spp. were the proteolytic bacteria that were most frequently present, comprising 59% of the isolates. They were always the numerically dominant proteolytic bacteria isolated from three kinds of fertilizer treatments (yearly application of rice-straw compost and chemical fertilizer, yearly application of chemical fertilizer, and no fertilizer application) and at three different stages of rice development (vegetative growth stage, maximal tillering stage, and harvest stage). Of the 411 proteolytic bacteria isolated, 124 isolates had stronger proteolytic activity than others on the basis of gelatin liquefaction tests and most of them were Bacillus spp. (100% in 1989 and 92.4% in 1991). Bacillus subtilis and Bacillus cereus were the main bacteria of this group and Bacillus mycoides, Bacillus licheniformis, and Bacillus megaterium were also present. We conclude that these Bacillus spp. are the primary source of soil protease in these paddy fields.

  1. Effect of proteolytic starter cultures as leavening agents of pizza dough.

    PubMed

    Pepe, O; Villani, F; Oliviero, D; Greco, T; Coppola, S

    2003-08-01

    Lactic acid bacteria (LAB) and yeasts were selected on the basis of in vitro proteolytic activity against wheat gluten protein and then assayed as leavening agents for pizza dough. Trials were carried out to compare a proteolytic starter (Prt(+)), consisting of Lactobacillus sakei T56, Weissella paramesenteroides A51 and Candida krusei G271, and a non-proteolytic starter (Prt(-)), consisting of Lb. sakei T58, W. paramesenteroides A58 and Saccharomyces cerevisiae T22. The proteolytic activity of the starter cultures was monitored immediately after mixing of the dough and throughout the fermentation process. The proteolytic activity was assessed by analysing the salt-soluble protein (SSP) and the dioxane-soluble protein (DSP) fractions of the pizza dough by discontinuous SDS-PAGE. Only the Prt(+) starter exhibited considerable qualitative and quantitative changes in the electrophoretic patterns of the protein fractions extracted. After the fermentation, the Prt(+) and Prt(-) doughs were tested to evaluate the influence of the proteolytic activity on the mechanical properties of the dough before and after baking. Indications emerged suggesting an influence of the proteolytic activity on the viscoelasticity of pizza dough. The pizza dough with Prt(+) strains showed an increase in viscous properties during the fermentation as compared with the Prt(-) dough. Moreover, an increase in the firmness of the crumb was observed in Prt(+) baked pizza dough.

  2. Sirtuins and Proteolytic Systems: Implications for Pathogenesis of Synucleinopathies

    PubMed Central

    Sampaio-Marques, Belém; Ludovico, Paula

    2015-01-01

    Insoluble and fibrillar forms of α-synuclein are the major components of Lewy bodies, a hallmark of several sporadic and inherited neurodegenerative diseases known as synucleinopathies. α-Synuclein is a natural unfolded and aggregation-prone protein that can be degraded by the ubiquitin-proteasomal system and the lysosomal degradation pathways. α-Synuclein is a target of the main cellular proteolytic systems, but it is also able to alter their function further, contributing to the progression of neurodegeneration. Aging, a major risk for synucleinopathies, is associated with a decrease activity of the proteolytic systems, further aggravating this toxic looping cycle. Here, the current literature on the basic aspects of the routes for α-synuclein clearance, as well as the consequences of the proteolytic systems collapse, will be discussed. Finally, particular focus will be given to the sirtuins’s role on proteostasis regulation, since their modulation emerged as a promising therapeutic strategy to rescue cells from α-synuclein toxicity. The controversial reports on the potential role of sirtuins in the degradation of α-synuclein will be discussed. Connection between sirtuins and proteolytic systems is definitely worth of further studies to increase the knowledge that will allow its proper exploration as new avenue to fight synucleinopathies. PMID:25946078

  3. The dependence on temperature and pH of the effects of zinc and copper on proteolytic activities of the digestive tract mucosa in piscivorous fish and their potential preys.

    PubMed

    Kuz'mina, V V; Ushakova, N V

    2010-09-01

    The dependence of the effects of zinc and copper on the activities of proteinases of the stomach and intestinal mucosa on temperature and pH in four species of boreal piscivorous fish (pike Esox lucius, zander Zander lucioperca, perch Perca fluviatilis and burbot Lota lota) as well as in some of their potential preys (kilka Clupeonella cultriventris, ruff Gymnocephalus cernuus, perch and roach Rutilus rutilus) was investigated. Species-specific differences of the effects of these heavy metals upon the activities of proteinases depending on temperature and pH were demonstrated. It was revealed that the stomach mucosa proteinases were more tolerant to the effects of the studied factors than the intestinal mucosa proteinases, especially true for pike. The effects of the heavy metals on the whole body proteinases of the fishes' potential preys were mostly dependent on temperature than on pH. At pH 3.0, the negative action of zinc and copper on the fish digestive tract mucosa proteolytic activity to a considerable degree was compensated by the high activity of the hemoglobinlytic proteinases, probably, cathepsine D.

  4. Inhibition of the hemorrhagic and proteolytic activities of Lansberg's hognose pit viper (Porthidium lansbergii hutmanni) venom by opossum (Didelphis marsupialis) serum: isolation of Didelphis marsupialis 0.15Dm fraction on DEAE-cellulose chromatography.

    PubMed

    Pineda, María E; Girón, María E; Estrella, Amalid; Sánchez, Elda E; Aguilar, Irma; Fernandez, Irma; Vargas, Alba M; Scannone, Héctor; Rodríguez-Acosta, Alexis

    2008-01-01

    Earlier studies have revealed the ability of sera from several mammals to neutralize the toxic effects of snake venom. The Venezuelan opossum (Didelphis marsupialis) is one that has been found to inhibit hemorrhagic and proteolytic activities of venoms from many species of snakes. In this article it is shown that the opossum sera and its 0.15DM fraction were able to completely neutralize both hemorrhagic and hydrolysis (proteolysis) of casein effects induced by venom of the Lansberg's hognose pit viper (Porthidium lansbergii hutmanni). We have used DEAE-cellulose ion exchange chromatography to collect protein fractions from D. marsupialis sera which were able to defend mice from the lethal effects of P.l. hutmanni venom. The fractions separated were homogeneous by conventional electrophoresis using SDS-PAGE. The protein bands obtained contained molecular weights of approximately 6 to 220 kDa. These results revealed the presence of proteases inhibitors in the opossum sera fractions and the inhibition of venom activity by opossum sera suggesting a reciprocal adaptation at the molecular level.

  5. Alternative Proteolytic Processing of Hepatocyte Growth Factor during Wound Repair

    PubMed Central

    Buchstein, Nils; Hoffmann, Daniel; Smola, Hans; Lang, Sabina; Paulsson, Mats; Niemann, Catherin; Krieg, Thomas; Eming, Sabine A.

    2009-01-01

    Wound healing is a crucial regenerative process in all organisms. We examined expression, integrity, and function of the proteins in the hepatocyte growth factor (HGF)/c-Met signaling pathway in normally healing and non-healing human skin wounds. Whereas in normally healing wounds phosphorylation of c-Met was most prominent in keratinocytes and dermal cells, in non-healing wounds phosphorylation of c-Met was barely detectable, suggesting reduced c-Met activation. In wound exudates obtained from non-healing, but not from healing wounds, HGF protein was a target of substantial proteolytic processing that was different from the classical activation by known serine proteases. Western blot analysis and protease inhibitor studies revealed that HGF is a target of neutrophil elastase and plasma kallikrein during skin repair. Proteolytic processing of HGF by each of these proteases significantly attenuated keratinocyte proliferation, wound closure capacity in vitro, and c-Met signal transduction. Our findings reveal a novel pathway of HGF processing during skin repair. Conditions in which proteases are imbalanced and tend toward increased proteolytic activity, as in chronic non-healing wounds, might therefore compromise HGF activity due to the inactivation of the HGF protein and/or the generation of HGF fragments that ultimately mediate a dominant negative effect and limit c-Met activation. PMID:19389925

  6. The N- and C-terminal autolytic fragments of CAPN3/p94/calpain-3 restore proteolytic activity by intermolecular complementation

    PubMed Central

    Ono, Yasuko; Shindo, Mayumi; Doi, Naoko; Kitamura, Fujiko; Gregorio, Carol C.

    2014-01-01

    CAPN3/p94/calpain-3, a calpain protease family member predominantly expressed in skeletal muscle, possesses unusually rapid and exhaustive autolytic activity. Mutations in the human CAPN3 gene impairing its protease functions cause limb-girdle muscular dystrophy type 2A (LGMD2A); yet, the connection between CAPN3’s autolytic activity and the enzyme’s function in vivo remain unclear. Here, we demonstrated that CAPN3 protease activity was reconstituted by intermolecular complementation (iMOC) between its two autolytic fragments. Furthermore, the activity of full-length CAPN3 active-site mutants was surprisingly rescued through iMOC with autolytic fragments containing WT amino acid sequences. These results provide evidence that WT CAPN3 can be formed by the iMOC of two different complementary CAPN3 mutants. The finding of iMOC-mediated restoration of calpain activity indicates a novel mechanism for the genotype–phenotype links in LGMD2A. PMID:25512505

  7. Structure and Functional Properties of the Active Form of the Proteolytic Complex, ClpP1P2, from Mycobacterium tuberculosis*

    PubMed Central

    Li, Mi; Kandror, Olga; Akopian, Tatos; Dharkar, Poorva; Wlodawer, Alexander; Maurizi, Michael R.; Goldberg, Alfred L.

    2016-01-01

    The ClpP protease complex and its regulatory ATPases, ClpC1 and ClpX, in Mycobacterium tuberculosis (Mtb) are essential and, therefore, promising drug targets. The Mtb ClpP protease consists of two heptameric rings, one composed of ClpP1 and the other of ClpP2 subunits. Formation of the enzymatically active ClpP1P2 complex requires binding of N-blocked dipeptide activators. We have found a new potent activator, benzoyl-leucine-leucine (Bz-LL), that binds with higher affinity and promotes 3–4-fold higher peptidase activity than previous activators. Bz-LL-activated ClpP1P2 specifically stimulates the ATPase activity of Mtb ClpC1 and ClpX. The ClpC1P1P2 and ClpXP1P2 complexes exhibit 2–3-fold enhanced ATPase activity, peptide cleavage, and ATP-dependent protein degradation. The crystal structure of ClpP1P2 with bound Bz-LL was determined at a resolution of 3.07 Å and with benzyloxycarbonyl-Leu-Leu (Z-LL) bound at 2.9 Å. Bz-LL was present in all 14 active sites, whereas Z-LL density was not resolved. Surprisingly, Bz-LL adopts opposite orientations in ClpP1 and ClpP2. In ClpP1, Bz-LL binds with the C-terminal leucine side chain in the S1 pocket. One C-terminal oxygen is close to the catalytic serine, whereas the other contacts backbone amides in the oxyanion hole. In ClpP2, Bz-LL binds with the benzoyl group in the S1 pocket, and the peptide hydrogen bonded between parallel β-strands. The ClpP2 axial loops are extended, forming an open axial channel as has been observed with bound ADEP antibiotics. Thus occupancy of the active sites of ClpP allosterically alters sites on the surfaces thereby affecting the association of ClpP1 and ClpP2 rings, interactions with regulatory ATPases, and entry of protein substrates. PMID:26858247

  8. [Influence of coordination compounds of germanium (IV) and stannum (IV) on activity of some microbial enzymes with glycolytic and proteolytic action].

    PubMed

    Varbanets', L D; Matseliukh, O V; Nidialkova, N A; Hudzenko, O V; Avdiiuk, K V; Shmatkova, N V; Seĭfullina, I Ĭ

    2014-01-01

    Influence of coordinative compounds of germanium (IV) and stanum (IV) (complexes of germanium (IV) with nicotinamide (Nad) [GeCl2(Nad)4]Cl2 (1) and complexes of stanum (IV) with 2-hydroxybenzoilhydrazone 4-dimetylaminobenzaldehide (2-OH-HBdb) [SnCl4(2-OH-Bdb-H)] (2), 3-hydroxy-2-naphtoilhydrazone 2-hydroxynaphtaldehide (3-OH-H2Lnf) [SnCl3(3-OH-HLnf)] (3) and izonicotinoilhydrazone 2-hydroxyibenzaldehide [SnCl3 (Is·H)] (4) on activity of peptidases 1 and 2 Bacillus thuringiensis, α-L-rhamnosidase Cryptococcus albidus, Eupenicillium erubescens and α-amylase Aspergillus flavus var. oryzae. Results testify that all studied compounds differ on their influence on activity of the enzymes tested: significantly don't change elastolytic activity of peptidases 1 and 2 B. thuringiensis, completely inhibit A. flavus var. oryzae amylase, activate or oppress of α-L-rhamnosidase C. albidus and E. erubescens. Considerable differences in compounds (3, 4) on activity observed in case of the last. It's possible that peculiarity of influence (1) in compare with (2-4) is connected with existence of different central atoms of complexants: germanium (IV) (1) and stanum (IV) (2-4). A certain analogy in oppression of C. albidus α-L-rhamnosidase by compounds (1) and (4) can explain with presence of a pyridinic ring at molecules of their ligands. The less activsty displayed compound (2) with coordinative knot {SnCl4ON}. Nature of compounds (3, 4) activity was absolutely different: essential increase of activity of C. albidus α-L-rhamnosidase and full oppression of E. erubescens α-L-rhamnosidase by compound (3), while the action of compound (4) was feed back. Taking into account identical coordination knot {SnCl3O2N} the major role in this case play change of a hydrazide fragment in molecules of their ligands.

  9. [Proteolytic control of the antirestriction activity of Tn2l, Tn5053, Tn5045 Tn501 TN402 non-conjugative transposons].

    PubMed

    Zavilgelsky, G B; Kotova, V Yu; Melkina, O E; Balabanov, V P; Mindlin, S Z

    2015-01-01

    Conjugative plasmids and conjugative transposons contain the genes, which products specifically inhibit the type I restriction--modification systems. Here is shown that non-conjugative transposons Tn2l, Tn5053, Tn5045, Tn501, Tn402 partially inhibit the restriction activity of the type I restriction-modification endonuclease EcoKI (R2M2S1) in Escherichia coli cells K12 (the phenomenon of restriction alleviation, RA). Antirestriction activity of the transposons is determined by the MerR and ArdD proteins. Antirestriction activity of transposons is absent in mutants E. coli K12 clpX and clpP and is decreased in mutants E. coli K12 recA, recBC and dnaQ (mutD). Induction of the RA in response to the MerR and ArdD activities is consistent with the production of unmodified target sequences following DNA repair and DNA synthesis associated with recombination repair or replication errors. RA effect is determined by the ClpXP-dependent degradation of the endonuclease activity R subunit of EcoKI (R2M2S1).

  10. Pepsinogens and pepsins from largemouth bass, Micropterus salmoides: purification and characterization with special reference to high proteolytic activities of bass enzymes.

    PubMed

    Miura, Yoko; Kageyama, Takashi; Moriyama, Akihiko

    2015-05-01

    Six pepsinogens were purified from the gastric mucosa of largemouth bass (Micropterus salmoides) by DEAE-Sephacel chromatography, Sephadex G-100 gel filtration, and Mono Q FPLC. The potential specific activities of two major pepsinogens, PG1-1 and PG2-2, against hemoglobin were 51 and 118 units/mg protein, respectively. The activity of pepsin 2-2 was the highest among the pepsins reported to date; this might be linked to the strongly carnivorous diet of the largemouth bass. The molecular masses of PG1-1 and PG2-2 were 39.0 and 41.0 kDa, respectively. The N-terminal amino acid sequences of PG1-1 and PG2-2 were LVQVPLEVGQTAREYLE- and LVRLPLIVGKTARQALLE-, respectively, showing similarities with those of fish type-A pepsinogens. The optimal pHs for hemoglobin-digestive activity of pepsins 1-1 and 2-2 were around 1.5 and 2.0, respectively, though both pepsins retained considerable activity at pHs over 3.5. They showed maximal activity around 50 and 40 °C, respectively. They were inhibited by pepstatin similarly to porcine pepsin A. The cleavage specificities clarified with oxidized insulin B chain were shown to be restricted to a few bonds consisting of hydrophobic/aromatic residues, such as the Leu(15)-Tyr(16), Phe(24)-Phe(25) and Phe(25)-Tyr(26) bonds. When hemoglobin was used as a substrate, the kcat/Km value of bass pepsin 2-2 was 4.6- to 36.8-fold larger than those of other fish pepsins. In the case of substance P, an ideal pepsin substrate mimic, the kcat/Km values were about 200-fold larger than those of porcine pepsin A, supporting the high activity of the bass pepsin.

  11. Proteolytic processing of myostatin is auto-regulated during myogenesis.

    PubMed

    McFarlane, Craig; Langley, Brett; Thomas, Mark; Hennebry, Alex; Plummer, Erin; Nicholas, Gina; McMahon, Chris; Sharma, Mridula; Kambadur, Ravi

    2005-07-01

    Myostatin, a potent negative regulator of myogenesis, is proteolytically processed by furin proteases into active mature myostatin before secretion from myoblasts. Here, we show that mature myostatin auto-regulates its processing during myogenesis. In a cell culture model of myogenesis, Northern blot analysis revealed no appreciable change in myostatin mRNA levels between proliferating myoblasts and differentiated myotubes. However, Western blot analysis confirmed a relative reduction in myostatin processing and secretion by differentiated myotubes as compared to proliferating myoblasts. Furthermore, in vivo results demonstrate a lower level of myostatin processing during fetal muscle development when compared to postnatal adult muscle. Consequently, high levels of circulatory mature myostatin were detected in postnatal serum, while fetal circulatory myostatin levels were undetectable. Since Furin proteases are important for proteolytically processing members of the TGF-beta superfamily, we therefore investigated the ability of myostatin to control the transcription of furin and auto-regulate the extent of its processing. Transfection experiments indicate that mature myostatin indeed regulates furin protease promoter activity. Based on these results, we propose a mechanism whereby myostatin negatively regulates its proteolytic processing during fetal development, ultimately facilitating the differentiation of myoblasts by controlling both furin protease gene expression and subsequent active concentrations of mature myostatin peptide.

  12. Evaluation of a Triple-Helical Peptide with Quenched Fluorophores for Optical Imaging of MMP-2 and MMP-9 Proteolytic Activity

    PubMed Central

    Zhang, Xuan; Bresee, Jamee; Cheney, Philip P.; Xu, Baogang; Bhowmick, Manishabrata; Cudic, Mare; Fields, Gregg B.; Edwards, Wilson Barry

    2015-01-01

    Matrix metalloproteinases (MMP) 2 and 9, the gelatinases, have consistently been associated with tumor progression. The development of gelatinase-specific probes will be critical for identifying in vivo gelatinoic activity to understand the molecular role of the gelatinases in tumor development. Recently, a self-assembling homotrimeric triple-helical peptide (THP), incorporating a sequence from type V collagen, with high substrate specificity to the gelatinases has been developed. To determine whether this THP would be suitable for imaging protease activity, 5-carboxyfluorescein (5FAM) was conjugated, resulting in 5FAM3-THP and 5FAM6-THP, which were quenched up to 50%. 5FAM6-THP hydrolysis by MMP-2 and MMP-9 displayed kcat/KM values of 1.5 × 104 and 5.4 × 103 M−1 s−1, respectively. Additionally 5FAM6-THP visualized gelatinase activity in gelatinase positive HT-1080 cells, but not in gelatinase negative MCF-7 cells. Furthermore, the fluorescence in the HT-1080 cells was greatly attenuated by the addition of a MMP-2 and MMP-9 inhibitor, SB-3CT, indicating that the observed fluorescence release was mediated by gelatinase proteolysis and not non-specific proteolysis of the THPs. These results demonstrate that THPs fully substituted with fluorophores maintain their substrate specificity to the gelatinases in human cancer cells and may be useful in in vivo molecular imaging of gelatinase activity. PMID:24959683

  13. Evaluation of a triple-helical peptide with quenched FluorSophores for optical imaging of MMP-2 and MMP-9 proteolytic activity.

    PubMed

    Zhang, Xuan; Bresee, Jamee; Cheney, Philip P; Xu, Baogang; Bhowmick, Manishabrata; Cudic, Mare; Fields, Gregg B; Edwards, Wilson Barry

    2014-06-23

    Matrix metalloproteinases (MMP) 2 and 9, the gelatinases, have consistently been associated with tumor progression. The development of gelatinase-specific probes will be critical for identifying in vivo gelatinoic activity to understand the molecular role of the gelatinases in tumor development. Recently, a self-assembling homotrimeric triple-helical peptide (THP), incorporating a sequence from type V collagen, with high substrate specificity to the gelatinases has been developed. To determine whether this THP would be suitable for imaging protease activity, 5-carboxyfluorescein (5FAM) was conjugated, resulting in 5FAM3-THP and 5FAM6-THP, which were quenched up to 50%. 5FAM6-THP hydrolysis by MMP-2 and MMP-9 displayed kcat/KM values of 1.5 × 104 and 5.4 × 103 M-1 s-1, respectively. Additionally 5FAM6-THP visualized gelatinase activity in gelatinase positive HT-1080 cells, but not in gelatinase negative MCF-7 cells. Furthermore, the fluorescence in the HT-1080 cells was greatly attenuated by the addition of a MMP-2 and MMP-9 inhibitor, SB-3CT, indicating that the observed fluorescence release was mediated by gelatinase proteolysis and not non-specific proteolysis of the THPs. These results demonstrate that THPs fully substituted with fluorophores maintain their substrate specificity to the gelatinases in human cancer cells and may be useful in in vivo molecular imaging of gelatinase activity.

  14. Target recognition by EcoKI: the recognition domain is robust and restriction-deficiency commonly results from the proteolytic control of enzyme activity.

    PubMed

    O'Neill, M; Powell, L M; Murray, N E

    2001-03-30

    We report a genetic and biochemical analysis of a target recognition domain (TRD) of EcoKI, a type I restriction and modification enzyme. The TRDs of type I R-M systems are within the specificity subunit (HsdS) and HsdS confers sequence specificity to a complex endowed with both restriction and modification activities. Random mutagenesis has revealed that most substitutions within the amino TRD of EcoKI, a region comprising 157 amino acid residues, have no detectable effect on the phenotype of the bacterium, even when the substitutions are non- conservative. The structure of the TRD appears to be robust. All but one of the six substitutions that confer a restriction-deficient, modification-deficient (r(-)m(-)) phenotype were found to be in the interval between residues 80 and 110, a region predicted by sequence comparisons to form part of the protein-DNA interface. Additional site-directed mutations affecting this interval commonly impair both restriction and modification. However, we show that an r(-) phenotype cannot be taken as evidence that the EcoKI complex lacks endonuclease activity; in response to even a slightly impaired modification efficiency, the endonuclease activity of EcoKI is destroyed by a process dependent upon the ClpXP protease. Enzymes from mutants with an r(-)m(-) phenotype commonly retain some sequence-specific activity; methylase activity can be detected on hemimethylated DNA substrates and residual endonuclease activity is implied whenever the viability of the r(-)m(-) bacterium is dependent on ClpXP. Conversely, the viability of ClpX(-) r(-)m(-) bacteria can be used as evidence for little, or no, endonuclease activity. Of 14 mutants with an r(-)m(-) phenotype, only six are viable in the absence of ClpXP. The significance of four of the six residues (G91, G105, F107 and G141) is enhanced by the finding that even conservative substitutions for these residues impair modification, thereby conferring an r(-)m(-) phenotype. Copyright 2001

  15. Anisi stellati fructus extract attenuates the in vitro and in vivo metastatic and angiogenic potential of malignant cancer cells by downregulating proteolytic activity and pro-angiogenic factors.

    PubMed

    Kim, Aeyung; Im, Minju; Ma, Jin Yeul

    2014-11-01

    Anisi stellati fructus (ASF), commonly known as star anise, has long been used as a traditional Chinese medicine to treat inflammation, nervousness, insomnia and pain. In recent studies, it has been demonstrated that ASF possesses anti-bacterial, anti-fungal and anti-oxidant activities, as well as exhibits inhibitory effects on capillary‑like tube formation in human umbilical vein endothelial cells (HUVECs). However, the effects of ASF extract on the metastatic potential of malignant tumor cells have not been examined. In this study, we found that daily oral administration of ASF (50 mg/kg) remarkably reduced the number of pulmonary metastatic colonies of B16F10 cells in C57BL/6J mice with no observed systemic toxicity. In an in vitro system, ASF inhibited metastatic properties, including anchorage‑independent colony formation, migration and invasion. Upon phorbol 12-myristate 13-acetate (PMA) stimulation, the mRNA levels of matrix metalloproteinases (MMPs) -9, -13, -14 and urokinase plasminogen activator (uPA) decreased in a dose-dependent manner with ASF treatment. Gelatinase, type I collagenase, and uPA activities were also suppressed efficiently by ASF treatment. In response to PMA, NF-κB and AP-1 activation as well as p38 phosphorylation, which are crucial for MMP activation, were significantly decreased by ASF. In particular, ASF considerably inhibited tumor-induced HUVEC migration and tube formation and suppressed in vivo tumor-induced angiogenesis via a reduction of pro-angiogenic factors in tumors. These results collectively indicate that ASF might be useful in the management of metastatic malignant tumors.

  16. Proteolytic activity in cowshed dust extracts induces C5a release in murine bronchoalveolar lavage fluids which may account for its protective properties in allergic airway inflammation.

    PubMed

    Stiehm, Matthias; Bufe, Albrecht; Peters, Marcus

    2013-01-01

    Intranasal application of cowshed dust extract (CDE) during sensitisation in a murine model of experimental asthma leads to a significant alleviation of the clinical parameters of the allergic immune response. However, neither the immunological mechanisms underlying this protective effect nor all of the protective substances included in CDE have yet been described. Recently, complement factor 5a (C5a) receptor signalling has been identified to play a regulatory role in allergic airway disease. Thus we investigated whether CDE can activate the complement system to release biologically active C5a in the lung. Proteins included in CDE were identified by mass spectrometry. Complement cleaving activity of a serine protease identified in CDE was validated with the purified enzyme, and the biological activity of the released C5a was determined. C5a was applied in a murine model of allergy to prove its protective impact on allergic airway disease. CDE induced the release of C5a in murine bronchoalveolar lavages (BAL). We identified a serine protease from the midgut of tenebrio molitor larvae in CDEs which was able to induce the release of biologically active C5a in murine BAL. We applied C5a in different doses to female Balb/c mice during the sensitisation phase and during the first antigen challenge and showed that C5a has the ability to dampen important parameters of allergic airway inflammation, such as infiltration of proinflammatory cells into lung tissue or Th2 cytokine secretion by lung cells. We conclude that the C5a generating enzyme included in CDE might account for some of the allergy protective effects of CDE by generation of C5a in murine lungs.

  17. Cell-layer-associated proteolytic cleavage of the telopeptides of type I collagen in fibroblast culture.

    PubMed Central

    Bateman, J F; Pillow, J J; Mascara, T; Medvedec, S; Ramshaw, J A; Cole, W G

    1987-01-01

    In human skin fibroblast cultures a fraction of the procollagen that was processed to collagen and remained in the cell layer was further proteolytically modified by removal of both N- and C-terminal telopeptides. The proteolytic activity was associated with the cell layer, since secreted collagens were found always to contain intact telopeptides. The inclusion of neutral polymers, which caused the accumulation of the collagen in the cell layer [Bateman, Cole, Pillow & Ramshaw (1986) J. Biol. Chem. 261, 4198-4203], made the telopeptide cleavage more apparent in those cells which expressed the proteolytic activity. The extent of this cleavage was variable from cell culture to cell culture and between experiments with the same fibroblast line. The proteolytic activity was pH-dependent; cleavage was greatest at a culture-medium pH of 7.5 and 8.0 and was completely inhibited at a culture-medium pH of 7.0 and 6.5. The activity was significantly inhibited by soybean trypsin inhibitor, an elastase-specific inhibitor (N-acetylalanylalanylprolylvalylchloromethane) and the thrombin inhibitor hirudin. This cell-associated proteolytic activity may play a role in collagen degradation by removing the telopeptides, which are the primary sites of collagen cross-linking, thus destabilizing the collagen matrix sufficiently to render it susceptible to further proteolytic breakdown. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:3311034

  18. The Androgen-Regulated Protease TMPRSS2 Activates aProteolytic Cascade Involving Components of the Tumor Microenvironment and Promotes Prostate Cancer Metastasis

    PubMed Central

    Lucas, Jared M.; Heinlein, Cynthia; Kim, Tom; Hernandez, Susana A.; Malik, Muzdah S.; True, Lawrence D.; Morrissey, Colm; Corey, Eva; Montgomery, Bruce; Mostaghel, Elahe; Clegg, Nigel; Coleman, Ilsa; Brown, Christopher M.; Schneider, Eric L.; Craik, Charles; Simon, Julian; Bedalov, Tony; Nelson, Peter S.

    2014-01-01

    TMPRSS2 is an androgen-regulated cell surface serine protease expressed predominantly in prostate epithelium. TMPRSS2 is expressed highly in localized high-grade prostate cancers and in the majority of human prostate cancer metastasis. Through the generation of mouse models with a targeted deletion of Tmprss2, we demonstrate that the activity of this protease regulates cancer cell invasion and metastasis to distant organs. By screening combinatorial peptide libraries we identified a spectrum of TMPRSS2 substrates that include pro-hepatocyte growth factor (HGF). HGF activated by TMPRSS2 promoted c-Met receptor tyrosine kinase signaling, and initiated a pro-invasive EMT phenotype. Chemical library screens identified a potent bioavailable TMPRSS2 inhibitor that suppressed prostate cancer metastasis in vivo. Together, these findings provide a mechanistic link between androgen-regulated signaling programs and prostate cancer metastasis that operate via context-dependent interactions with extracellular constituents of the tumor microenvironment. PMID:25122198

  19. [The effect of x-ray radiation and hypoxia on the proteolytic activity of the normal rat spleen and during tumor growth].

    PubMed

    Likholat, E A; Reva, A D; Sokolov, I I; Chernaia, V I

    1991-01-01

    The influence of X-radiation on activity of lysosomal enzymes (D, L, H cathepsins) in rat spleen tissue and in inoculated rat sarcoma 45 has been investigated. Intact rats and rats with tumors were subjected to whole-body and sarcoma 45 to local irradiation with doses of 0.155 C/kg and 0.31 C/kg in conditions of breathing gas hypoxic mixture containing 90% of nitrogen and 10% of oxygen (GHM-10). The combined exposure to radiation and GHM-10 was shown to produce a certain protective action (e.g. normalized cathepsin activity) in the spleen. In the tumor tissue the protective effect of GHM-10 was absent.

  20. Characterization of a heat-resistant extracellular protease from Pseudomonas fluorescens 07A shows that low temperature treatments are more effective in deactivating its proteolytic activity.

    PubMed

    Alves, Maura P; Salgado, Rafael L; Eller, Monique R; Vidigal, Pedro Marcus P; Fernandes de Carvalho, Antonio

    2016-10-01

    This work discusses the biological and biochemical characterization of an extracellular protease produced by Pseudomonas fluorescens. The enzyme has a molecular weight of 49.486 kDa and hydrolyzes gelatin, casein, and azocasein, but not BSA. Its maximum activity is found at 37°C and pH 7.5, but it retained almost 70% activity at pH 10.0. It was shown to be a metalloprotease inhibited by Cu(2+), Ni(2+), Zn(2+), Hg(2+), Fe(2+), and Mg(2+), but induced by Mn(2+). After incubation at 100°C for 5min, the enzyme presented over 40% activity, but only 14 to 30% when submitted to milder heat treatments. This behavior may cause significant problems under conditions commonly used for the processing and storage of milk and dairy products, particularly UHT milk. A specific peptide sequenced by mass spectrometer analysis allowed the identification of gene that encodes this extracellular protease in the genome of Pseudomonas fluorescens 07A strain. The enzyme has 477 AA and highly conserved Ca(2+)- and Zn(2+)-binding domains, indicating that Ca(2+), the main ion in milk, is also a cofactor. This work contributes to the understanding of the biochemical aspects of enzyme activity and associates them with its sequence and structure. These findings are essential for the full understanding and control of these enzymes and the technological problems they cause in the dairy industry. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  1. Purification by cobalamin-Sepharose affinity chromatography and intrinsic factor-binding activity of an extramembrane proteolytic product from pig ileal mucosa.

    PubMed Central

    Yerima, A; Safi, A; Gastin, I; Michalski, J C; Saunier, M; Gueant, J L

    1996-01-01

    We have purified a cobalamin-binding protein obtained by papain digestion of pig intestine by cobalamin-AH-Sepharose affinity chromatography, with a purification factor of 17,300, a yield of 63% and a cobalamin-binding activity of 11,260 pmol/mg of protein. The protein contained 3.8% carbohydrate and was O- and N-glycosylated. Its molecular mass was 69 kDa on SDS/PAGE and its isoelectric point was 5.1. It had a binding activity for both [57Co]cobalamin and [57Co]cobalamin-intrinsic factor in native PAGE autoradiography and it inhibited the binding of intrinsic factor to the intact intestinal receptor with an IC50 of 49.31 nmol/l in a radioisotope assay. In conclusion, the purified protein shared a binding activity for both cobalamin and intrinsic factor-cobalamin complexes and could correspond to the extracellular domain of the ileal intrinsic factor receptor. PMID:8573109

  2. The androgen-regulated protease TMPRSS2 activates a proteolytic cascade involving components of the tumor microenvironment and promotes prostate cancer metastasis.

    PubMed

    Lucas, Jared M; Heinlein, Cynthia; Kim, Tom; Hernandez, Susana A; Malik, Muzdah S; True, Lawrence D; Morrissey, Colm; Corey, Eva; Montgomery, Bruce; Mostaghel, Elahe; Clegg, Nigel; Coleman, Ilsa; Brown, Christopher M; Schneider, Eric L; Craik, Charles; Simon, Julian A; Bedalov, Antonio; Nelson, Peter S

    2014-11-01

    TMPRSS2 is an androgen-regulated cell-surface serine protease expressed predominantly in prostate epithelium. TMPRSS2 is expressed highly in localized high-grade prostate cancers and in the majority of human prostate cancer metastases. Through the generation of mouse models with a targeted deletion of Tmprss2, we demonstrate that the activity of this protease regulates cancer cell invasion and metastasis to distant organs. By screening combinatorial peptide libraries, we identified a spectrum of TMPRSS2 substrates that include pro-hepatocyte growth factor (HGF). HGF activated by TMPRSS2 promoted c-MET receptor tyrosine kinase signaling, and initiated a proinvasive epithelial-to-mesenchymal transition phenotype. Chemical library screens identified a potent bioavailable TMPRSS2 inhibitor that suppressed prostate cancer metastasis in vivo. Together, these findings provide a mechanistic link between androgen-regulated signaling programs and prostate cancer metastasis that operate via context-dependent interactions with extracellular constituents of the tumor microenvironment. The vast majority of prostate cancer deaths are due to metastasis. Loss of TMPRSS2 activity dramatically attenuated the metastatic phenotype through mechanisms involving the HGF-c-MET axis. Therapeutic approaches directed toward inhibiting TMPRSS2 may reduce the incidence or progression of metastasis in patients with prostate cancer. ©2014 American Association for Cancer Research.

  3. The Proteolytic Activation of (H3N2) Influenza A Virus Hemagglutinin Is Facilitated by Different Type II Transmembrane Serine Proteases

    PubMed Central

    Kühn, Nora; Bergmann, Silke; Kösterke, Nadine; Lambertz, Ruth L. O.; Keppner, Anna; van den Brand, Judith M. A.; Weiß, Siegfried; Hummler, Edith; Hatesuer, Bastian

    2016-01-01

    ABSTRACT Cleavage of influenza virus hemagglutinin (HA) by host cell proteases is necessary for viral activation and infectivity. In humans and mice, members of the type II transmembrane protease family (TTSP), e.g., TMPRSS2, TMPRSS4, and TMPRSS11d (HAT), have been shown to cleave influenza virus HA for viral activation and infectivity in vitro. Recently, we reported that inactivation of a single HA-activating protease gene, Tmprss2, in knockout mice inhibits the spread of H1N1 influenza viruses. However, after infection of Tmprss2 knockout mice with an H3N2 influenza virus, only a slight increase in survival was observed, and mice still lost body weight. In this study, we investigated an additional trypsin-like protease, TMPRSS4. Both TMPRSS2 and TMPRSS4 are expressed in the same cell types of the mouse lung. Deletion of Tmprss4 alone in knockout mice does not protect them from body weight loss and death upon infection with H3N2 influenza virus. In contrast, Tmprss2−/− Tmprss4−/− double-knockout mice showed a remarkably reduced virus spread and lung pathology, in addition to reduced body weight loss and mortality. Thus, our results identified TMPRSS4 as a second host cell protease that, in addition to TMPRSS2, is able to activate the HA of H3N2 influenza virus in vivo. IMPORTANCE Influenza epidemics and recurring pandemics are responsible for significant global morbidity and mortality. Due to high variability of the virus genome, resistance to available antiviral drugs is frequently observed, and new targets for treatment of influenza are needed. Host cell factors essential for processing of the virus hemagglutinin represent very suitable drug targets because the virus is dependent on these host factors for replication. We reported previously that Tmprss2-deficient mice are protected against H1N1 virus infections, but only marginal protection against H3N2 virus infections was observed. Here we show that deletion of two host protease genes, Tmprss2 and

  4. PROTEOLYTIC CLEAVAGE OF VERSICAN DURING LIMB JOINT DEVELOPMENT

    PubMed Central

    Capehart, Anthony A.

    2011-01-01

    Versican is highly expressed in developing joint interzones during limb morphogenesis. The present study was undertaken to examine whether proteolytic cleavage of versican occurs that could potentially impact its function during the process of embryonic synovial joint formation. Using an antibody to the DPEAAE neoepitope generated by ADAMTS proteolysis, versican amino terminal cleavage fragments were detected in joint interzones at 12–16 days post coitum (dpc). ADAMTS-1 localization overlapped that of DPEAAE-reactive versican fragments suggesting it as one possible protease activity involved in processing of versican in the interzone. Results show that increased cleavage of versican in the interzone accompanies cavitation and suggests that proteolytic modification of versican may be important during the process of synovial joint maturation PMID:20101710

  5. Selective Substrates and Inhibitors for Kallikrein-Related Peptidase 7 (KLK7) Shed Light on KLK Proteolytic Activity in the Stratum Corneum.

    PubMed

    de Veer, Simon J; Furio, Laetitia; Swedberg, Joakim E; Munro, Christopher A; Brattsand, Maria; Clements, Judith A; Hovnanian, Alain; Harris, Jonathan M

    2017-02-01

    Proteases have pivotal roles in the skin's outermost layer, the epidermis. In the stratum corneum, serine proteases from the kallikrein-related peptidase (KLK) family have been implicated in several key homeostatic processes, including desquamation. However, the precise contribution of specific KLKs to each process remains unclear. To address this, we used a chemical biology approach and designed selective substrates and inhibitors for KLK7, the most abundant KLK protease in the stratum corneum. The resulting KLK7 inhibitor is the most potent inhibitor of this protease reported to date (Ki = 140 pM), and displays at least 1,000-fold selectivity over several proteases that are related by function (KLK5 and KLK14) or specificity (chymotrypsin). We then used substrates and inhibitors for KLK5, KLK7, and KLK14 to explore the activity of each protease in the stratum corneum using casein zymography and an ex vivo desquamation assay. These experiments provide the most detailed assessment of each KLK's contribution to corneocyte shedding in the plantar stratum corneum, revealing that inhibition of KLK7 alone is sufficient to block shedding, whereas KLK5 is also a major contributor. Collectively, these findings unveil chemical tools for studying KLK activity and demonstrate their potential for characterizing KLK biological functions in epidermal homeostasis. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Ubiquitination of E3 ligases: self-regulation of the ubiquitin system via proteolytic and non-proteolytic mechanisms

    PubMed Central

    de Bie, P; Ciechanover, A

    2011-01-01

    Ubiquitin modification of many cellular proteins targets them for proteasomal degradation, but in addition can also serve non-proteolytic functions. Over the last years, a significant progress has been made in our understanding of how modification of the substrates of the ubiquitin system is regulated. However, little is known on how the ubiquitin system that is comprised of ∼1500 components is regulated. Here, we discuss how the biggest subfamily within the system, that of the E3 ubiquitin ligases that endow the system with its high specificity towards the numerous substrates, is regulated and in particular via self-regulation mediated by ubiquitin modification. Ligases can be targeted for degradation in a self-catalyzed manner, or through modification mediated by an external ligase(s). In addition, non-proteolytic functions of self-ubiquitination, for example activation of the ligase, of E3s are discussed. PMID:21372847

  7. Proteolytic activity, mycotoxins and andrastin A in Penicillium roqueforti strains isolated from Cabrales, Valdeón and Bejes-Tresviso local varieties of blue-veined cheeses.

    PubMed

    Fernández-Bodega, M A; Mauriz, E; Gómez, A; Martín, J F

    2009-11-30

    High quality local varieties of blue-veined cheese are made in the villages of the valleys of Cabrales, Valdeón and Bejes-Tresviso in North Spain. Penicillium roqueforti strains have been isolated from each of those blue cheeses and compared with the collection strain P. roqueforti CECT 2905 (ATCC 10110) and a strain 'Valdeón-industrial' used for large scale production of Valdeón cheese. Using molecular genetics techniques and 5.8S and 18S rRNAs and the D1-D2 regions of 28S rRNA all strains were identified as authentic P. roqueforti. These strains from local varieties of blue cheese could be distinguished from the Valdeón-industrial strain and the control strain CECT 2905 by the mitochondrial DNA restriction pattern. The industrial strain showed high levels of aspartylprotease AspA, whereas the culture collection strain showed barely detectable levels of this enzyme, as shown by proteolysis tests and by immunodetection with anti-AspA antibodies. The lipolytic activity was similar in the strains isolated from the three types of local blue cheeses. The strains isolated from the local varieties of the blue cheese produced moderate levels of PR toxin, whereas the Valdeón-industrial strains showed a higher content of this mycotoxin. All strains (except the control strain CECT 2905) showed similar levels of roquefortine C. The antitumoral compound andrastin A was produced by all strains at different levels. P. roqueforti CECT 2905 showed high ability to synthesize this compound. Andrastin A was present in all industrial and local varieties of blue cheese. The content of andrastin A was similar to that of other well-known blue cheeses from France and Denmark.

  8. Proteolytic effect of Cynara cardunculus rennet for use in the elaboration of 'Torta del Casar' cheese.

    PubMed

    Ordiales, Elena; Benito, Maria José; Martin, Alberto; Fernández, Margarita; Hernández, Alejandro; de Guia Córdoba, Maria

    2013-11-01

    The purpose of this work was to analyse the influence of rennet from different Cynara cardunculus plants, selected for its clotting and proteolytic activity on caseins, on the characteristics of manufactured 'Torta del Casar' cheeses. After classifying the cardoon according to proteolytic activity into five groups of greater or lesser activity, 16 batches of cheeses were made with rennet derived from different wild cardoon plants. We observed a major development of the proteolysis during ripening leading to the generation of non-protein nitrogen compounds. Especially noteworthy was the relationship of amino acid nitrogen (AN) generation with rennet clotting activity after 24 h of maceration, and the fact that the production of biogenic amines was not related to the proteolytic activity of the rennet. The activities of the rennet observed 'in vitro' were also developed 'in vivo' in the cheeses, with the different rennets used affecting the final sensory characteristics of cheeses. The rennet with high clotting activity after 24 h of maceration was positively correlated with the creaminess, viscosity, and acceptability of the cheese. However, the high proteolytic activity rennet negatively influenced the acidity, bitterness, and creaminess parameters. Therefore the most appropriate cardoons for making this cheese are those with higher clotting activities and moderate proteolytic activities especially on β-casein. The use of controlled and characterised cardoons in the manufacturing process of Torta del Casar is fundamental to obtaining the homogeneous product demanded by the Torta del Casar Registry of the Protected Designation of Origin.

  9. Trophic Interactions of Proteolytic Bacteria Proteinivorax tanatarense in an Alkalinphilic Microbial Community.

    PubMed

    Boltyanskaya, Yu V; Kevbrin, V V

    2016-07-01

    Lythic action of an anaerobic proteolytic bacterium Proteinivorax tanatarense on organisms with different cell wall types was studied. In the absence of photosynthetic oxygen release, this proteolytic was able to grow on intact biomass of cyanobacteria belonging to various systematic groups. Itis probably their usual saprotrophic-satellite responsible for the regulation of abundance of primary producers during the dark phase. Growth also occurred on the biomass of a nonphototrophic gram-negative microorganism-Halomonas campisalis, a common component of alkaliphilic. microbial communities: Comparative analysis of the interaction of the proteolytic with.H. campisalis cells at different physiological states revealed the lytic action to be re- stricted to dead and/or weakened cells, rather than the actively dividing ones. Strict specificity of the action of the proteolytic bacterium on gram-negative microorganisms with no effect on gram-positive ones was shown.

  10. Organic solvent-tolerant bacterium which secretes an organic solvent-stable proteolytic enzyme

    SciTech Connect

    Ogino, Hiroyasu; Yasui, Kiyoshi; Shiotani, Takashi

    1995-12-01

    A bacterial strain which can be grown in a medium containing organic solvents and can secrete a proteolytic enzyme was isolated and identified as Pseudomonas aeruginosa. The strain was derived by the following two-step procedures: high proteolytic enzyme producers were first isolated by the usual method, and then the organic solvent-tolerant microorganism was selected from these high-rate proteolytic enzyme producers. The proteolytic activity of the supernatant of the culture was stable in the presence of various organic solvents. The stability of the enzyme in the presence of organic solvents, of which the values of the logarithm of the partition coefficient (log P) were equal to or more than 3.2, was almost the same as that in the absence of organic solvents. It is expected that both the solvent-tolerant microorganism and the solvent-stable enzyme produced by this strain can be used as catalysts for reactions in the presence of organic solvents.

  11. Chronic venous disease - Part II: Proteolytic biomarkers in wound healing.

    PubMed

    Ligi, Daniela; Mosti, Giovanni; Croce, Lidia; Raffetto, Joseph D; Mannello, Ferdinando

    2016-10-01

    Venous leg ulcers (VLU) are characterized by sustained proteolytic microenvironment impairing the healing process. Wound fluid (WF) reflect the biomolecular activities occurring within the wound area; however, it is unclear if WF from different healing phases have different proteolytic profiles and how VLU microenvironment affects the wound healing mechanisms. We investigated the proteolytic network of WF from distinct VLU phases, and in WF- and LPS-stimulated THP-1 monocytes treated with glycosaminoglycan sulodexide, a well known therapeutic approach for VLU healing. WF were collected from patients with VLU during inflammatory (Infl) and granulating (Gran) phases. WF and THP-1 supernatants were analyzed for nine matrix metalloproteinases (MMP) and four tissue inhibitors of metalloproteinases (TIMP) by multiplex immunoassays. Our results demonstrated that: 1) WF from Infl VLU contained significantly increased concentrations of MMP-2, MMP-9, MMP-12, TIMP-1, and TIMP-2 compared to Gran WF; 2) WF from Gran VLU showed significantly increased levels of MMP-1, MMP-7, MMP-13, and TIMP-4 compared to Infl WF; 3) LPS- and WF-stimulation of THP-1 cells significantly increased the expression of several MMP compared to untreated cells; 4) Sulodexide treatment of both LPS- and WF-stimulated THP-1 significantly down-regulated the release of several MMPs. Our study provides evidence-based medicine during treatment of patients with VLU. WF from Infl and Gran VLU have different MMP and TIMP signatures, consistent with their clinical state. The modulation of proteolytic pathways in wound microenvironment by glycosaminoglycan sulodexide, provide insights for translating research into clinical practice during VLU therapy. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. A review of statistical methods for prediction of proteolytic cleavage.

    PubMed

    duVerle, David A; Mamitsuka, Hiroshi

    2012-05-01

    A fundamental component of systems biology, proteolytic cleavage is involved in nearly all aspects of cellular activities: from gene regulation to cell lifecycle regulation. Current sequencing technologies have made it possible to compile large amount of cleavage data and brought greater understanding of the underlying protein interactions. However, the practical impossibility to exhaustively retrieve substrate sequences through experimentation alone has long highlighted the need for efficient computational prediction methods. Such methods must be able to quickly mark substrate candidates and putative cleavage sites for further analysis. Available methods and expected reliability depend heavily on the type and complexity of proteolytic action, as well as the availability of well-labelled experimental data sets: factors varying greatly across enzyme families. For this review, we chose to give a quick overview of the general issues and challenges in cleavage prediction methods followed by a more in-depth presentation of major techniques and implementations, with a focus on two particular families of cysteine proteases: caspases and calpains. Through their respective differences in proteolytic specificity (high for caspases, broader for calpains) and data availability (much lower for calpains), we aimed to illustrate the strengths and limitations of techniques ranging from position-based matrices and decision trees to more flexible machine-learning methods such as hidden Markov models and Support Vector Machines. In addition to a technical overview for each family of algorithms, we tried to provide elements of evaluation and performance comparison across methods.

  13. Ectopically expressed pro-group X secretory phospholipase A2 is proteolytically activated in mouse adrenal cells by furin-like proprotein convertases: implications for the regulation of adrenal steroidogenesis.

    PubMed

    Layne, Joseph D; Shridas, Preetha; Webb, Nancy R

    2015-03-20

    Group X secretory phospholipase A2 (GX sPLA2) hydrolyzes mammalian cell membranes, liberating free fatty acids and lysophospholipids. GX sPLA2 is produced as a pro-enzyme (pro-GX sPLA2) that contains an N-terminal 11-amino acid propeptide ending in a dibasic motif, suggesting cleavage by a furin-like proprotein convertase (PC). Although propeptide cleavage is clearly required for enzymatic activity, the protease(s) responsible for pro-GX sPLA2 activation have not been identified. We previously reported that GX sPLA2 negatively regulates adrenal glucocorticoid production, likely by suppressing liver X receptor-mediated activation of steroidogenic acute regulatory protein expression. In this study, using a FLAG epitope-tagged pro-GX sPLA2 expression construct (FLAG-pro-GX sPLA2), we determined that adrenocorticotropic hormone (ACTH) enhanced FLAG-pro-GX sPLA2 processing and phospholipase activity secreted by Y1 adrenal cells. ACTH increased the expression of furin and PCSK6, but not other members of the PC family, in Y1 cells. Overexpression of furin and PCSK6 in HEK 293 cells significantly enhanced FLAG-pro-GX sPLA2 processing, whereas siRNA-mediated knockdown of both PCs almost completely abolished FLAG-pro-GX sPLA2 processing in Y1 cells. Expression of either furin or PCSK6 enhanced the ability of GX sPLA2 to suppress liver X receptor reporter activity. The PC inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone significantly suppressed FLAG-pro-GX sPLA2 processing and sPLA2 activity in Y1 cells, and it significantly attenuated GX sPLA2-dependent inhibition of steroidogenic acute regulatory protein expression and progesterone production. These findings provide strong evidence that pro-GX sPLA2 is a substrate for furin and PCSK6 proteolytic processing and define a novel mechanism for regulating corticosteroid production in adrenal cells.

  14. A non-proteolytic function of ubiquitin in transcription repression

    PubMed Central

    Ndoja, Ada; Yao, Tingting

    2014-01-01

    Regulation of transcription is vitally important for maintaining normal cellular homeostasis and is also the basis for cellular differentiation, morphogenesis and the adaptability of any organism. Transcription activators, which orchestrate time and locus-specific assembly of complex transcription machinery, act as key players in these processes. One way in which these activators are controlled is by the covalent attachment of the conserved protein, ubiquitin (Ub), which can serve as either a proteolytic or non-proteolytic signal. For a subset of the activators, polyubiquitination-dependent degradation of the activator controls its abundance. In these cases transcription activation can require protein synthesis as well as internal or external stimulus. In contrast, other activators have been reported to undergo mono- or oligoubiquitination that does not lead to protein degradation. The mechanisms by which monoubiquitination of transcription activators affect their activities have been poorly understood. In a recent study, we demonstrated that monoubiquitination of some transcription activators can inhibit transcription by recruiting the AAA+ ATPase Cdc48 (also known in metazoan organisms as p97 or valosin-contain protein, VCP), which then extracts the ubiquitinated activator from DNA. PMID:28357251

  15. Isolation, subunit structure, and proteolytic modification of bovine factor VIII.

    PubMed

    Legaz, M E; Weinstein, M J; Heldebrant, C M; Davie, E W

    1975-01-20

    A new method has been described for the isolation of factor VIII. The method results in a high yield of factor VIII that is homogeneous by several different criteria. The purified protein is very stable and is not dissociated in the presence of 1 M NaCl or 0.25 M CaCl2. The highly purified protein is readily activated and inactivated by various proteolytic enzymes, such as thrombin, plasmin, and trypsin. The molecular events that lead to the activation reaction, however, have not been established.

  16. Tissue kallikrein proteolytic cascade pathways in normal physiology and cancer.

    PubMed

    Pampalakis, Georgios; Sotiropoulou, Georgia

    2007-09-01

    Human tissue kallikreins (KLKs or kallikrein-related peptidases) are a subgroup of extracellular serine proteases that act on a wide variety of physiological substrates, while they display aberrant expression patterns in certain types of cancer. Differential expression patterns lead to the exploitation of these proteins as new cancer biomarkers for hormone-dependent malignancies, in particular. The prostate-specific antigen or kallikrein-related peptidase 3 (PSA/KLK3) is an established tumor marker for the diagnosis and monitoring of prostate cancer. It is well documented that specific KLK genes are co-expressed in tissues and in various pathologies suggesting their participation in complex proteolytic cascades. Here, we review the currently established knowledge on the involvement of KLK proteolytic cascades in the regulation of physiological and pathological processes in prostate tissue and in skin. It is well established that the activity of KLKs is often regulated by auto-activation and subsequent autolytic internal cleavage leading to enzymatic inactivation, as well as by inhibitory serpins or by allosteric inhibition by zinc ions. Redistribution of zinc ions and alterations in their concentration due to physiological or pathological reasons activates specific KLKs initiating the kallikrein cascade(s). Recent studies on kallikrein substrate specificity allowed for the construction of a kallikrein interaction network involved in semen liquefaction and prostate cancer, as well as in skin pathologies, such as skin desquamation, psoriasis and cancer. Furthermore, we discuss the crosstalks between known proteolytic pathways and the kallikrein cascades, with emphasis on the activation of plasmin and its implications in prostate cancer. These findings may have clinical implications for the underlying molecular mechanism and management of cancer and other disorders in which KLK activity is elevated.

  17. A Comparative Study of New Aspergillus Strains for Proteolytic Enzymes Production by Solid State Fermentation

    PubMed Central

    Ortiz, Gastón Ezequiel; Noseda, Diego Gabriel; Ponce Mora, María Clara; Recupero, Matías Nicolás; Blasco, Martín; Albertó, Edgardo

    2016-01-01

    A comparative study of the proteolytic enzymes production using twelve Aspergillus strains previously unused for this purpose was performed by solid state fermentation. A semiquantitative and quantitative evaluation of proteolytic activity were carried out using crude enzymatic extracts obtained from the fermentation cultures, finding seven strains with high and intermediate level of protease activity. Biochemical, thermodynamics, and kinetics features such as optimum pH and temperature values, thermal stability, activation energy (Ea), quotient energy (Q10), Km, and Vmax were studied in four enzymatic extracts from the selected strains that showed the highest productivity. Additionally, these strains were evaluated by zymogram analysis obtaining protease profiles with a wide range of molecular weight for each sample. From these four strains with the highest productivity, the proteolytic extract of A. sojae ATCC 20235 was shown to be an appropriate biocatalyst for hydrolysis of casein and gelatin substrates, increasing its antioxidant activities in 35% and 125%, respectively. PMID:26989505

  18. A Comparative Study of New Aspergillus Strains for Proteolytic Enzymes Production by Solid State Fermentation.

    PubMed

    Ortiz, Gastón Ezequiel; Noseda, Diego Gabriel; Ponce Mora, María Clara; Recupero, Matías Nicolás; Blasco, Martín; Albertó, Edgardo

    2016-01-01

    A comparative study of the proteolytic enzymes production using twelve Aspergillus strains previously unused for this purpose was performed by solid state fermentation. A semiquantitative and quantitative evaluation of proteolytic activity were carried out using crude enzymatic extracts obtained from the fermentation cultures, finding seven strains with high and intermediate level of protease activity. Biochemical, thermodynamics, and kinetics features such as optimum pH and temperature values, thermal stability, activation energy (E a), quotient energy (Q 10), K m , and V max were studied in four enzymatic extracts from the selected strains that showed the highest productivity. Additionally, these strains were evaluated by zymogram analysis obtaining protease profiles with a wide range of molecular weight for each sample. From these four strains with the highest productivity, the proteolytic extract of A. sojae ATCC 20235 was shown to be an appropriate biocatalyst for hydrolysis of casein and gelatin substrates, increasing its antioxidant activities in 35% and 125%, respectively.

  19. Protease inhibitors and proteolytic signalling cascades in insects.

    PubMed

    Gubb, David; Sanz-Parra, Arantza; Barcena, Laura; Troxler, Laurent; Fullaondo, Ane

    2010-12-01

    Proteolytic signalling cascades control a wide range of physiological responses. In order to respond rapidly, protease activity must be maintained at a basal level: the component zymogens must be sequentially activated and actively degraded. At the same time, signalling cascades must respond precisely: high target specificity is required. The insects have a wide range of trapping- and tight-binding protease inhibitors, which can regulate the activity of individual proteases. In addition, the interactions between component proteases of a signalling cascade can be modified by serine protease homologues. The suicide-inhibition mechanism of serpin family inhibitors gives rapid turnover of both protease and inhibitor, but target specificity is inherently broad. Similarly, the TEP/macroglobulins have extremely broad target specificity, which suits them for roles as hormone transport proteins and sensors of pathogenic virulence factors. The tight-binding inhibitors, on the other hand, have a lock-and-key mechanism capable of high target specificity. In addition, proteins containing multiple tight-binding inhibitory domains may act as scaffolds for the assembly of signalling complexes. Proteolytic cascades regulated by combinations of different types of inhibitor could combine the rapidity of suicide-inhibitors with the specificity lock-and-key inhibitors. This would allow precise control of physiological responses and may turn out to be a general rule.

  20. Expression and proteolytic processing of mammalian purple acid phosphatase in CHO-K1 cells.

    PubMed

    Wang, Yunling; Andersson, Göran

    2007-05-01

    Rat recombinant purple acid phosphatase (PAP) stably expressed in fibroblast-like CHO-K1 cells was purified and characterized with respect to post-translational modifications such as N-glycosylation and proteolytic processing in order to elucidate subcellular and molecular pathways for proteolytic activation. In these cells, proteolytically processed PAP was more abundant than the monomeric form. PAP-transfected CHO-K1 cells were expressing active cathepsin K intracellularly, which was partially co-localized with PAP. However, neither cathepsin K nor trypsin digestion of the purified monomeric PAP in vitro did result in a two-subunit form with kinetic and electrophoretic properties resembling the endogenous cellular two-subunit form. Treatment of PAP-transfected CHO-K1 cells with the cysteine proteinase inhibitor E-64 suggested that only a minor fraction of secreted PAP is processed intracellularly by cysteine proteinases. These data do not support a dominant or critical role for cathepsins or trypsin-like serine proteinases in the proteolytic activation of PAP in CHO-K1 cells, implicating yet unidentified proteinases in the proteolytic processing of both intracellular and secreted PAP in this cell line.

  1. The proteolytic system of lactic acid bacteria.

    PubMed

    Mayo, B

    1993-12-01

    Lactic acid bacteria are widely used throughout the world, empirically or deliberately, in the manufacturing of several food and feed stuffs, including milk products (such as cheese, butter, yoghurt, buttermilk, etc.), fermented vegetables (pickles, olives and sauerkraut), sausages, sourdough bread and silage, due to their ability to convert sugars into lactic acid. Of these, dairy products are of outstanding economic importance. Starter cultures used in the dairy industry are mixtures of carefully selected lactic acid bacteria which are added to the milk to fulfil the desired fermentation. Dairy starter cultures must reach high densities in milk in order to produce lactic acid at the required rates for manufacturing. Under these conditions, amino acids supply becomes limitant due to their scarce concentration in milk and to the auxotrophies shown by many starter bacteria. This implies the necessity of a proteolytic system, able to degrade the most abundant protein in milk, casein, into assimilable amino acids and peptides. Casein degradation and utilization require the concerted action of proteinases, peptidases and amino acid and peptide uptake systems. This whole set of enzymes constitutes the proteolytic system. In this article an overview of the recent biochemical and genetic data on the proteolytic system of lactic acid bacteria will be presented.

  2. Proteolytic crosstalk in multi-protease networks

    NASA Astrophysics Data System (ADS)

    Ogle, Curtis T.; Mather, William H.

    2016-04-01

    Processive proteases, such as ClpXP in E. coli, are conserved enzyme assemblies that can recognize and rapidly degrade proteins. These proteases are used for a number of purposes, including degrading mistranslated proteins and controlling cellular stress response. However, proteolytic machinery within the cell is limited in capacity and can lead to a bottleneck in protein degradation, whereby many proteins compete (‘queue’) for proteolytic resources. Previous work has demonstrated that such queueing can lead to pronounced statistical relationships between different protein counts when proteins compete for a single common protease. However, real cells contain many different proteases, e.g. ClpXP, ClpAP, and Lon in E. coli, and it is not clear how competition between proteins for multiple classes of protease would influence the dynamics of cellular networks. In the present work, we theoretically demonstrate that a multi-protease proteolytic bottleneck can substantially couple the dynamics for both simple and complex (oscillatory) networks, even between substrates with substantially different affinities for protease. For these networks, queueing often leads to strong positive correlations between protein counts, and these correlations are strongest near the queueing theoretic point of balance. Furthermore, we find that the qualitative behavior of these networks depends on the relative size of the absolute affinity of substrate to protease compared to the cross affinity of substrate to protease, leading in certain regimes to priority queue statistics.

  3. Characterization of the proteolytic system present in Vasconcellea quercifolia latex.

    PubMed

    Torres, María José; Trejo, Sebastián Alejandro; Obregón, Walter David; Avilés, Francesc Xavier; López, Laura María Isabel; Natalucci, Claudia Luisa

    2012-11-01

    Vasconcellea quercifolia (Caricaceae) latex contains several cysteine endopeptidases with high proteolytic activity. Cysteine endopeptidases are the main active compounds used by the plant as a defense mechanism. A proteolytic preparation from V. quercifolia ("oak leaved papaya") latex was purified by cation exchange chromatography. From SDS-PAGE and blotting of the selected fractions, the N-terminal amino acid sequences of polypeptides were determined by Edman's degradation. The analysis by peptide mass fingerprinting (PMF) of the enzymes allowed their characterization and confirmed the presence of seven different cysteine proteinases in the latex of V. quercifolia. Moreover, the comparison between the tryptic maps with those deposited in databases using the MASCOT tool showed that none of the isolated proteases matched with another plant protease. Notably, a propeptidase was detected in the plant latex, which is being the first report in this sense. Furthermore, the cDNA of one of the cysteine proteases that is expressed in the latex of V. quercifolia was cloned and sequenced. The consensus sequence was aligned using the ClustalX web server, which allowed detecting a high degree of identity with cysteine proteases of the Caricaceae family and establishing the evolutionary relationship between them. We also observed a high conservation degree for those amino acid residues which are essential for the catalytic activity and tridimensional structure of the plant proteases belonging to the subfamily C1A. The PMF analysis strongly suggests that the sequence obtained corresponds to the VQ-III peptidase.

  4. Effect of ethylene producer ethrel and antioxidant ionol (BHT) on the proteolytic apparatus in coleoptiles of wheat seedlings during apoptosis.

    PubMed

    Fedoreyeva, L I; Aleksandrushkina, N I; Dunaevsky, Ya E; Belozersky, M A; Vanyushin, B F

    2003-04-01

    It was established that total proteolytic activity in etiolated wheat seedlings changes in ontogenesis in cycles: peaks of proteolytic activity correspond to the 3rd, 5th, and 8th days of seedling growth, respectively. The maximum of proteolytic activity preceded the maximum of nuclease activity, which may be due to activation of nucleases by proteolytic enzymes. According to inhibitory analysis the cysteine and serine proteases play the main role in apoptosis in wheat coleoptiles. Growing of seedlings in the presence of ethrel stimulated apoptosis in the coleoptile, and it increased (almost 6-fold) the proteolytic activity in its cells. On the other hand, the antioxidant ionol (BHT) suppressed the induction of proteases, particularly at the second stage of coleoptile development, and it slowed down the increase in the nuclease activity after 6th day of the seedling life. It is suggested that phytohormones and antioxidants participate in regulation of apoptosis in the ageing coleoptile, directly or indirectly effecting the proteolytic apparatus in the coleoptile cells.

  5. Modulation of the proteolytic cascade systems by high dose corticosteroids.

    PubMed

    Aasen, A O; Ruud, T E; Pillgram-Larsen, J; Röise, O; Stadaas, J

    1985-01-01

    The effects of high-dose corticosteroids (HDC) on activities within the proteolytic cascade systems were studied in vitro and in vivo using chromogenic peptide substrate assays. In in vitro experiments 20 mg methylprednisolone sodium succinate (Solu-Medrol) per ml plasma significantly inhibited activation of plasma prekallikrein, prothrombin and plasminogen and reduced functional plasma kallikrein inhibition, antithrombin and antiplasmin activities. The effects of HDC on activities within these proteolytic cascade systems were further evaluated in experimental acute pancreatitis in pigs. Acute pancreatitis was induced by injection of Na-taurocholate into the pancreatic duct. Seven test animals received methylprednisolone sodium succinate 30 mg per kg intravenously for 30 minutes before the induction of pancreatitis as pretreatment. Eight animals remained untreated. Trypsin (TRY), plasma prekallikrein (PKK), plasma kallikrein (KK) and functional plasma kallikrein inhibition capacity (KKI) were studied in the peritoneal exudate. Cardiac output (CO) and mean arterial pressure (MAP) were monitored regularly before and during a 6 hour observation period. During untreated pancreatitis a reduction of PKK levels of about 40% were found, paralleled by an increased KK activity and a reduction of KKI capacity. Several of the animals experienced high TRY activities. The mortality rate was 63% (5 out of 8 animals). In the pretreated groups, all animals survived the observation period. CO and MAP were significantly less reduced than the untreated group at 6 hours. HDC was also found to reduce significantly plasma kallikrein activities in the peritoneal exudate compared with untreated animals. No changes in TRY activities were found in pretreated animals. Furthermore, plasma prekallikrein and functional plasma kallikrein inhibition values in the exudate were elevated significantly in HDC treated animals compared with untreated animals.

  6. The structure and proteolytic processing of Cbln1 complexes.

    PubMed

    Bao, Dashi; Pang, Zhen; Morgan, James I

    2005-11-01

    The hexadecapeptide cerebellin is present in the brains of many vertebrate species and is derived from a larger protein, Cbln1 (cerebellin 1 precursor protein). Although cerebellin has features of a neuropeptide, Cbln1 belongs to the C1q/tumor necrosis factor superfamily of secreted proteins, suggesting that it is the biologically active molecule and the proteolytic events that generate cerebellin serve another function. Therefore, we assessed whether Cbln1 undergoes proteolytic processing and determined what consequences the cleavage events necessary to produce cerebellin have on the structure of Cbln1. Substantial degradation of Cbln1 was evident in the synaptic compartment of cerebellum and lysates of cultured cerebellar neurons and cells transfected with Cbln1 expression vectors. However, only uncleaved Cbln1 containing the cerebellin motif was released and assembled into hexameric complexes. Using yeast two hybrid and mammalian expression systems we show that the cleavages required to produce cerebellin influence the subunit stoichiometry of Cbln1 complexes. Cleavage at the N-terminus of the cerebellin sequence in Cbln1 yields trimeric complexes by separating the trimer-mediating C-terminal C1q domain from conserved N-terminal cysteine residues that mediate higher order oligomerization. Cleavage at the C-terminus of the cerebellin motif disrupts the C1q domain and abolishes subunit interactions. Functional implications of these data are discussed.

  7. Matriptase-2 (TMPRSS6): a proteolytic regulator of iron homeostasis

    PubMed Central

    Ramsay, Andrew J.; Hooper, John D.; Folgueras, Alicia R.; Velasco, Gloria; López-Otín, Carlos

    2009-01-01

    Maintaining the body’s levels of iron within precise boundaries is essential for normal physiological function. Alterations of these levels below or above the healthy limit lead to a systemic deficiency or overload in iron. The type-two transmembrane serine protease (TTSP), matriptase-2 (also known as TMPRSS6), is attracting significant amounts of interest due to its recently described role in iron homeostasis. The finding of this regulatory role for matriptase-2 was originally derived from the observation that mice deficient in this protease present with anemia due to elevated levels of hepcidin and impaired intestinal iron absorption. Further in vitro analysis has demonstrated that matriptase-2 functions to suppress bone morphogenetic protein stimulation of hepcidin transcription through cell surface proteolytic processing of the bone morphogenetic protein co-receptor hemojuvelin. Consistently, the anemic phenotype of matriptase-2 knockout mice is mirrored in humans with matripase-2 mutations. Currently, 14 patients with iron-refractory iron deficiency anemia (IRIDA) have been reported to harbor various genetic mutations that abrogate matriptase-2 proteolytic activity. In this review, after overviewing the membrane anchored serine proteases, in particular the TTSP family, we summarize the identification and characterization of matriptase-2 and describe its functional relevance in iron metabolism. PMID:19377077

  8. The caspase 3-dependent apoptotic effect of pycnogenol in human oral squamous cell carcinoma HSC-3 cells

    PubMed Central

    Yang, In-Hyoung; Shin, Ji-Ae; Kim, Lee-Han; Kwon, Ki Han; Cho, Sung-Dae

    2016-01-01

    In the present study, the apoptotic effect of pycnogenol and its molecular mechanism in human oral squamous cell carcinoma HSC-3 cells were investigated. Pycnogenol significantly inhibited the viability of HSC-3 cells and suppressed neoplastic cell transformation in HSC-3 cells and TPA-treated JB6 cells. It caused caspase-dependent apoptosis evidenced by the increase in cleaved poly (ADP-ribose) polymerase and caspase 3 in a dose-dependent manner. Pycnogenol increased Bak protein by enhancing its protein stability whereas other Bcl-2 family members were not altered. In addition, the treatment with pycnogenol led to the production of reactive oxygen species and N-acetyl-l-cysteine almost blocked pycnogenol-induced reactive oxygen species generation. Taken together, these findings suggest that pycnogenol may be a potential candidate for the chemoprevention or chemotherapy of human oral cancer. PMID:26798196

  9. The caspase 3-dependent apoptotic effect of pycnogenol in human oral squamous cell carcinoma HSC-3 cells.

    PubMed

    Yang, In-Hyoung; Shin, Ji-Ae; Kim, Lee-Han; Kwon, Ki Han; Cho, Sung-Dae

    2016-01-01

    In the present study, the apoptotic effect of pycnogenol and its molecular mechanism in human oral squamous cell carcinoma HSC-3 cells were investigated. Pycnogenol significantly inhibited the viability of HSC-3 cells and suppressed neoplastic cell transformation in HSC-3 cells and TPA-treated JB6 cells. It caused caspase-dependent apoptosis evidenced by the increase in cleaved poly (ADP-ribose) polymerase and caspase 3 in a dose-dependent manner. Pycnogenol increased Bak protein by enhancing its protein stability whereas other Bcl-2 family members were not altered. In addition, the treatment with pycnogenol led to the production of reactive oxygen species and N-acetyl-l-cysteine almost blocked pycnogenol-induced reactive oxygen species generation. Taken together, these findings suggest that pycnogenol may be a potential candidate for the chemoprevention or chemotherapy of human oral cancer.

  10. Mate (Ilex paraguariensis St. Hilaire) saponins induce caspase-3-dependent apoptosis in human colon cancer cells in vitro

    USDA-ARS?s Scientific Manuscript database

    Saponins are naturally occurring metabolites present in Yerba mate (Ilex paraguariensis), and other plant sources which have been associated with several health benefits. Mate saponins were extracted with methanol from dry leaves, partially purified and quantified. UV-HPLC analysis showed that the m...

  11. Specific proteolytic fragmentation of p60v-src in transformed cell lysates.

    PubMed Central

    Wells, S K; Collett, M S

    1983-01-01

    Work involving the transforming protein, p60v-src, of Rous sarcoma virus has resulted in the extensive characterization of its protein structure and associated phosphotransferase activity. However, in many investigations proteolytic fragments (principally p52v-src) of the src protein are actually studied. Here, we emphasize potential problems in the interpretation of experimental results in which the proteolytic fragmentation of p60v-src may be involved and offer several means for the complete prevention of this p60v-src degradation. Images PMID:6306278

  12. The proteolytic system of lactic acid bacteria revisited: a genomic comparison

    PubMed Central

    2010-01-01

    Background Lactic acid bacteria (LAB) are a group of gram-positive, lactic acid producing Firmicutes. They have been extensively used in food fermentations, including the production of various dairy products. The proteolytic system of LAB converts proteins to peptides and then to amino acids, which is essential for bacterial growth and also contributes significantly to flavor compounds as end-products. Recent developments in high-throughput genome sequencing and comparative genomics hybridization arrays provide us with opportunities to explore the diversity of the proteolytic system in various LAB strains. Results We performed a genome-wide comparative genomics analysis of proteolytic system components, including cell-wall bound proteinase, peptide transporters and peptidases, in 22 sequenced LAB strains. The peptidase families PepP/PepQ/PepM, PepD and PepI/PepR/PepL are described as examples of our in silico approach to refine the distinction of subfamilies with different enzymatic activities. Comparison of protein 3D structures of proline peptidases PepI/PepR/PepL and esterase A allowed identification of a conserved core structure, which was then used to improve phylogenetic analysis and functional annotation within this protein superfamily. The diversity of proteolytic system components in 39 Lactococcus lactis strains was explored using pangenome comparative genome hybridization analysis. Variations were observed in the proteinase PrtP and its maturation protein PrtM, in one of the Opp transport systems and in several peptidases between strains from different Lactococcus subspecies or from different origin. Conclusions The improved functional annotation of the proteolytic system components provides an excellent framework for future experimental validations of predicted enzymatic activities. The genome sequence data can be coupled to other "omics" data e.g. transcriptomics and metabolomics for prediction of proteolytic and flavor-forming potential of LAB strains

  13. Cellulolytic and proteolytic ability of bacteria isolated from gastrointestinal tract and composting of a hippopotamus.

    PubMed

    da Cruz Ramos, Geomárcia Feitosa; Ramos, Patricia Locosque; Passarini, Michel Rodrigo Zambrano; Vieira Silveira, Marghuel A; Okamoto, Débora Noma; de Oliveira, Lilian Caroline Gonçalves; Zezzo, Larissa Vieira; Marem, Alyne; Santos Rocha, Rafael Costa; da Cruz, João Batista; Juliano, Luiz; de Vasconcellos, Suzan Pantaroto

    2016-03-01

    The bioprospection for cellulase and protease producers is a promise strategy for the discovery of potential biocatalysts for use in hydrolysis of lignocellulosic materials as well as proteic residues. These enzymes can increment and turn viable the production of second generation ethanol from different and alternative sources. In this context, the goal of this study was the investigation of cellulolytic and proteolytic abilities of bacteria isolated from the gastrointestinal tract of a hippopotamus as well as from its composting process. It is important to highlight that hippopotamus gastrointestinal samples were a non-typical sources of efficient hydrolytic bacteria with potential for application in biotechnological industries, like biofuel production. Looking for this, a total of 159 bacteria were isolated, which were submitted to qualitative and quantitative enzymatic assays. Proteolytic analyzes were conducted through the evaluation of fluorescent probes. Qualitative assays for cellulolytic abilities revealed 70 positive hits. After quantitative analyzes, 44 % of these positive hits were selected, but five (5) strains showed cellulolytic activity up to 11,8 FPU/mL. Regarding to proteolytic activities, six (6) strains showed activity above 10 %, which overpassed results described in the literature. Molecular analyzes based on the identification of 16S rDNA, revealed that all the selected bacterial isolates were affiliated to Bacillus genus. In summary, these results strongly indicate that the isolated bacteria from a hippopotamus can be a potential source of interesting biocatalysts with cellulolytic and proteolytic activities, with relevance for industrial applications.

  14. Interaction of Leptospira interrogans with human proteolytic systems enhances dissemination through endothelial cells and protease levels.

    PubMed

    Vieira, Monica L; Alvarez-Flores, Miryam P; Kirchgatter, Karin; Romero, Eliete C; Alves, Ivy J; de Morais, Zenaide M; Vasconcellos, Silvio A; Chudzinski-Tavassi, Ana M; Nascimento, Ana L T O

    2013-05-01

    We have recently reported the ability of Leptospira to capture plasminogen (PLG) and generate plasmin (PLA) bound on the microbial surface in the presence of exogenous activators. In this work, we examined the effects of leptospiral PLG binding for active penetration through the endothelial cell barrier and activation. The results indicate that leptospires with PLG association or PLA activation have enhanced migration activity through human umbilical vein endothelial cell (HUVEC) monolayers compared with untreated bacteria. Leptospira cells coated with PLG were capable of stimulating the expression of PLG activators by HUVECs. Moreover, leptospires endowed with PLG or PLA promoted transcriptional upregulation matrix metalloprotease 9 (MMP-9). Serum samples from patients with confirmed leptospirosis showed higher levels of PLG activators and total MMP-9 than serum samples from normal (healthy) subjects. The highest level of PLG activators and total MMP-9 was detected with microscopic agglutination test (MAT)-negative serum samples, suggesting that this proteolytic activity stimulation occurs at the early stage of the disease. Furthermore, a gelatin zymography profile obtained for MMPs with serum samples from patients with leptospirosis appears to be specific to leptospiral infection because serum samples from patients with unrelated infectious diseases produced no similar degradation bands. Altogether, the data suggest that the Leptospira-associated PLG or PLA might represent a mechanism that contributes to bacterial penetration of endothelial cells through an activation cascade of events that enhances the proteolytic capability of the organism. To our knowledge, this is the first proteolytic activity associated with leptospiral pathogenesis described to date.

  15. Structural aberrations in T-even bacteriophage. VII. In vitro analysis of the canavanine-mediated inhibition of proteolytic cleavage.

    PubMed Central

    Bolin, R W; Cummings, D J

    1975-01-01

    Canavanine arrests a critical function in head morphogenesis and the potential for forming giant T-even phage particles termed lollipops is induced. Formation of the particles requires the addition of arginine and the restoration of normal functions. We now report on an investigation into the effects of canavanine on both the T4-induced proteolytic activity and on the substrate proteins. Using an in vitro cleavage assay we have shown that the gene 21-dependent proteolytic activity from canavanine-treated extracts is markedly inhibited, whereas the substrate proteins retain a high susceptibility for cleavage. The proteolytic activity in extracts treated with canavanine followed by arginine is readily detectable, and proteins previously synthesized in the presence of canavanine can be cleaved. Protein synthesis is apparently required for the appearance of the proteolytic activity after the canavanine-arginine treatment. Mixing experiments suggest the requirement for a component of the gene 21-dependent proteolytic activity that is not coded for by gene 21. Images PMID:1185853

  16. Organic Solvent-Tolerant Bacterium Which Secretes an Organic Solvent-Stable Proteolytic Enzyme

    PubMed Central

    Ogino, H.; Yasui, K.; Shiotani, T.; Ishihara, T.; Ishikawa, H.

    1995-01-01

    A bacterial strain which can be grown in a medium containing organic solvents and can secrete a proteolytic enzyme was isolated and identified as Pseudomonas aeruginosa. The strain was derived by the following two-step procedures: high proteolytic enzyme producers were first isolated by the usual method, and then the organic solvent-tolerant microorganism was selected from these high-rate proteolytic enzyme producers. The proteolytic activity of the supernatant of the culture was stable in the presence of various organic solvents. The stability of the enzyme in the presence of organic solvents, of which the values of the logarithm of the partition coefficient (log P) were equal to or more than 3.2, was almost the same as that in the absence of organic solvents. It is expected that both the solvent-tolerant microorganism and the solvent-stable enzyme produced by this strain can be used as catalysts for reactions in the presence of organic solvents. PMID:16535181

  17. Characterisation of a novel proteolytic enzyme localised to goblet cells in rat and man.

    PubMed Central

    Nexø, E; Poulsen, S S; Hansen, S N; Kirkegaard, P; Olsen, P S

    1984-01-01

    A proteolytic enzyme, ingobsin , purified from rat duodenal extracts is shown to be localised to intestinal goblet cells of both man and rat. Enzyme positive cells decrease in number from duodenum to colon. The enzyme is a 33 000 Mr protein with an isoelectric point of 5.1. The pH optimum for enzymatic activity is 7.4-8.0. Based on substrate specificity for arg-x, lys-x and to a lesser degree tyr-x, on the effect of diisopropylphosphorofluoride , Trasylol and phenylmethylsulfonylfluoride and on proteolytic activity towards intact proteins, ingobsin is classified as a serine proteinase with endoproteolytic activity. Images Fig. 2 Fig. 4 Fig. 6 PMID:6735249

  18. Research Applications of Proteolytic Enzymes in Molecular Biology

    PubMed Central

    Mótyán, János András; Tóth, Ferenc; Tőzsér, József

    2013-01-01

    Proteolytic enzymes (also termed peptidases, proteases and proteinases) are capable of hydrolyzing peptide bonds in proteins. They can be found in all living organisms, from viruses to animals and humans. Proteolytic enzymes have great medical and pharmaceutical importance due to their key role in biological processes and in the life-cycle of many pathogens. Proteases are extensively applied enzymes in several sectors of industry and biotechnology, furthermore, numerous research applications require their use, including production of Klenow fragments, peptide synthesis, digestion of unwanted proteins during nucleic acid purification, cell culturing and tissue dissociation, preparation of recombinant antibody fragments for research, diagnostics and therapy, exploration of the structure-function relationships by structural studies, removal of affinity tags from fusion proteins in recombinant protein techniques, peptide sequencing and proteolytic digestion of proteins in proteomics. The aim of this paper is to review the molecular biological aspects of proteolytic enzymes and summarize their applications in the life sciences. PMID:24970197

  19. Proteolytic inactivation of tissue factor pathway inhibitor by bacterial omptins

    PubMed Central

    Yun, Thomas H.; Cott, Jessica E.; Tapping, Richard I.; Slauch, James M.

    2009-01-01

    The immune response to infection includes activation of the blood clotting system, leading to extravascular fibrin deposition to limit the spread of invasive microorganisms. Some bacteria have evolved mechanisms to counteract this host response. Pla, a member of the omptin family of Gram-negative bacterial proteases, promotes the invasiveness of the plague bacterium, Yersinia pestis, by activating plasminogen to plasmin to digest fibrin. We now show that the endogenous anticoagulant tissue factor pathway inhibitor (TFPI) is also highly sensitive to proteolysis by Pla and its orthologs OmpT in Escherichia coli and PgtE in Salmonella enterica serovar Typhimurium. Using gene deletions, we demonstrate that bacterial inactivation of TFPI requires omptin expression. TFPI inactivation is mediated by proteolysis since Western blot analysis showed that TFPI cleavage correlated with loss of anticoagulant function in clotting assays. Rates of TFPI inactivation were much higher than rates of plasminogen activation, indicating that TFPI is a better substrate for omptins. We hypothesize that TFPI has evolved sensitivity to proteolytic inactivation by bacterial omptins to potentiate procoagulant responses to bacterial infection. This may contribute to the hemostatic imbalance in disseminated intravascular coagulation and other coagulopathies accompanying severe sepsis. PMID:18988866

  20. Intestinal Absorption of Fibrinolytic and Proteolytic Lumbrokinase Extracted from Earthworm, Eisenia andrei

    PubMed Central

    Yan, Xiang Mei; Kim, Chung-Hyo; Lee, Chul Kyu; Shin, Jang Sik; Cho, Il Hwan

    2010-01-01

    To investigate the intestinal absorption of a fibrinolytic and proteolytic lumbrokinase extracted from Eisenia andrei, we used rat everted gut sacs and an in situ closed-loop recirculation method. We extracted lumbrokinase from Eisenia andrei, and then raised polyclonal antibody against lumbrokinase. Fibrinolytic activity and proteolytic activity in the serosal side of rat everted gut sacs incubated with lumbrokinase showed dose- and time-dependent patterns. Immunological results obtained by western blotting serosal side solution using rat everted gut sacs method showed that lumbrokinase proteins between 33.6 and 54.7 kDa are absorbed mostly by the intestinal epithelium. Furthermore, MALDI-TOF mass spectrometric analysis of plasma fractions obtained by in situ recirculation method confirmed that lumbrokinase F1 is absorbed into blood. These results support the notion that lumbrokinase can be absorbed from mucosal lumen into blood by oral administration. PMID:20473377

  1. Scube2 enhances proteolytic Shh processing from the surface of Shh-producing cells.

    PubMed

    Jakobs, Petra; Exner, Sebastian; Schürmann, Sabine; Pickhinke, Ute; Bandari, Shyam; Ortmann, Corinna; Kupich, Sabine; Schulz, Philipp; Hansen, Uwe; Seidler, Daniela G; Grobe, Kay

    2014-04-15

    All morphogens of the Hedgehog (Hh) family are synthesized as dual-lipidated proteins, which results in their firm attachment to the surface of the cell in which they were produced. Thus, Hh release into the extracellular space requires accessory protein activities. We suggested previously that the proteolytic removal of N- and C-terminal lipidated peptides (shedding) could be one such activity. More recently, the secreted glycoprotein Scube2 (signal peptide, cubulin domain, epidermal-growth-factor-like protein 2) was also implicated in the release of Shh from the cell membrane. This activity strictly depended on the CUB domains of Scube2, which derive their name from the complement serine proteases and from bone morphogenetic protein-1/tolloid metalloproteinases (C1r/C1s, Uegf and Bmp1). CUB domains function as regulators of proteolytic activity in these proteins. This suggested that sheddases and Scube2 might cooperate in Shh release. Here, we confirm that sheddases and Scube2 act cooperatively to increase the pool of soluble bioactive Shh, and that Scube2-dependent morphogen release is unequivocally linked to the proteolytic processing of lipidated Shh termini, resulting in truncated soluble Shh. Thus, Scube2 proteins act as protease enhancers in this setting, revealing newly identified Scube2 functions in Hh signaling regulation.

  2. Porcine purple acid phosphatase: heterologous expression, characterization, and proteolytic analysis.

    PubMed

    Naseri, Joseph Itor; Truong, Ngoc Thanh; Hörentrup, Jens; Kuballa, Petric; Vogel, Andreas; Rompel, Annette; Spener, Friedrich; Krebs, Bernt

    2004-12-01

    Uteroferrin is an iron-binding glycoprotein, which is abundantly synthesized in porcine uterine glandular endometrium and believed to be involved in maternal/fetal iron transport. In the present study, uteroferrin has been cloned and functionally expressed using baculovirus-infected insect host cells Spodoptera frugiperda. The work also addresses the possible role of proteolytic cleavage to facilitate the release of uteroferrin-bound iron. The enzyme secreted in culture medium exhibits a molecular mass and catalytic properties similar to native porcine uteroferrin. The specific activity was estimated at 233 U/mg using p-nitrophenyl phosphate as substrate. Partial cleavage of the enzyme with trypsin resulted in a 1.7-fold enhancement in specific activity and a two-subunit polypeptide as observed in preparations of most mammalian purple acid phosphatases. Digestion with the aspartic protease pepsin resulted in a 2.5-fold enzyme inactivation correlated with the appearance of low molecular weight polypeptide fragments and the release of enzyme-bound iron.

  3. Proteolytic enzymes in seawater: contribution of prokaryotes and protists

    NASA Astrophysics Data System (ADS)

    Obayashi, Y.; Suzuki, S.

    2016-02-01

    Proteolytic enzyme is one of the major catalysts of microbial processing of organic matter in biogeochemical cycle. Here we summarize some of our studies about proteases in seawater, including 1) distribution of protease activities in coastal and oceanic seawater, 2) responses of microbial community and protease activities in seawater to organic matter amending, and 3) possible contribution of heterotrophic protists besides prokaryotes to proteases in seawater, to clarify cleared facts and remaining questions. Activities of aminopeptidases, trypsin-type and chymotrypsin-type proteases were detected from both coastal and oceanic seawater by using MCA-substrate assay. Significant activities were detected from not only particulate (cell-associated) fraction but also dissolved fraction of seawater, especially for trypsin-type and chymotrypsin-type proteases. Hydrolytic enzymes in seawater have been commonly thought to be mainly derived from heterotrophic prokaryotes; however, it was difficult to determine actual source organisms of dissolved enzymes in natural seawater. Our experiment with addition of dissolved protein to subtropical oligotrophic Pacific water showed drastically enhancement of the protease activities especially aminopeptidases in seawater, and the prokaryotic community structure simultaneously changed to be dominant of Bacteroidetes, indicating that heterotrophic bacteria were actually one of the sources of proteases in seawater. Another microcosm experiment with free-living marine heterotrophic ciliate Paranophrys marina together with an associated bacterium showed that extracellular trypsin-type activity was mainly attributed to the ciliate. The protist seemed to work in organic matter digestion in addition to be a grazer. From the results, we propose a system of organic matter digestion by prokaryotes and protists in aquatic environments, although their actual contribution in natural environments should be estimated in future studies.

  4. Proteolytic Systems in Milk: Perspectives on the Evolutionary Function within the Mammary Gland and the Infant

    PubMed Central

    Dallas, David C.; Murray, Niamh M.; Gan, Junai

    2015-01-01

    Milk contains elements of numerous proteolytic systems (zymogens, active proteases, protease inhibitors and protease activators) produced in part from blood, in part by mammary epithelial cells and in part by immune cell secretion. Researchers have examined milk proteases for decades, as they can cause major defects in milk quality and cheese production. Most previous research has examined these proteases with the aim to eliminate or control their actions. However, our recent peptidomics research demonstrates that these milk proteases produce specific peptides in healthy milk and continue to function within the infant’s gastrointestinal tract. These findings suggest that milk proteases have an evolutionary function in aiding the infant’s digestion or releasing functional peptides. In other words, the mother provides the infant with not only dietary proteins but also the means to digest them. However, proteolysis in the milk is controlled by a balance of protease inhibitors and protease activators so that only a small portion of milk proteins are digested within the mammary gland. This regulation presents a question: If proteolysis is beneficial to the infant, what benefits are gained by preventing complete proteolysis through the presence of protease inhibitors? In addition to summarizing what is known about milk proteolytic systems, we explore possible evolutionary explanations for this proteolytic balance. PMID:26179272

  5. Luciferase protection against proteolytic degradation: a key for improving signal in nano-system biology.

    PubMed

    Ataei, Farangis; Hosseinkhani, Saman; Khajeh, Khosro

    2009-10-26

    Luciferase is most widely used bioluminescence protein in biotechnological processes, but the enzyme is susceptible to proteolytic degradation, thereby its intracellular half-life decreased. Osmolytes are known to enhance the stability of proteins and protect them in a native folded and functional state. The effects of osmolytes, including sucrose, glycine and DMSO on the stability of luciferase were investigated. To different extents, all osmolytes protected the luciferase towards proteolytic degradation in a concentration-dependent manner. The results showed that 1.5M sucrose, 1.5M glycine and 15% DMSO are the best. The ability of these osmolytes to protect luciferase against proteolysis decreased from sucrose, glycine, and finally DMSO. Enzymatic kinetic data showed that the luciferase activity is significantly kept in the presence of sucrose and glycine compared to DMSO, particularly at high temperatures. Bioluminescence intensity, circular dichroism (CD), intrinsic and ANS fluorescence experiments showed change in secondary and tertiary luciferase structure. These results suggest that osmolytes exert an important effect on stabilization of luciferase conformation; decreasing the unfolding rate, preventing adaptation and binding of luciferase at the active site of proteases, thereby the proteolytic digestion reduced and its active conformation was kept.

  6. Proteolytic Systems in Milk: Perspectives on the Evolutionary Function within the Mammary Gland and the Infant.

    PubMed

    Dallas, David C; Murray, Niamh M; Gan, Junai

    2015-12-01

    Milk contains elements of numerous proteolytic systems (zymogens, active proteases, protease inhibitors and protease activators) produced in part from blood, in part by mammary epithelial cells and in part by immune cell secretion. Researchers have examined milk proteases for decades, as they can cause major defects in milk quality and cheese production. Most previous research has examined these proteases with the aim to eliminate or control their actions. However, our recent peptidomics research demonstrates that these milk proteases produce specific peptides in healthy milk and continue to function within the infant's gastrointestinal tract. These findings suggest that milk proteases have an evolutionary function in aiding the infant's digestion or releasing functional peptides. In other words, the mother provides the infant with not only dietary proteins but also the means to digest them. However, proteolysis in the milk is controlled by a balance of protease inhibitors and protease activators so that only a small portion of milk proteins are digested within the mammary gland. This regulation presents a question: If proteolysis is beneficial to the infant, what benefits are gained by preventing complete proteolysis through the presence of protease inhibitors? In addition to summarizing what is known about milk proteolytic systems, we explore possible evolutionary explanations for this proteolytic balance.

  7. Regulation of acetylation restores proteolytic function of diseased myocardium in mouse and human.

    PubMed

    Wang, Ding; Fang, Caiyun; Zong, Nobel C; Liem, David A; Cadeiras, Martin; Scruggs, Sarah B; Yu, Hongxiu; Kim, Allen K; Yang, Pengyuan; Deng, Mario; Lu, Haojie; Ping, Peipei

    2013-12-01

    Proteasome complexes play essential roles in maintaining cellular protein homeostasis and serve fundamental roles in cardiac function under normal and pathological conditions. A functional detriment in proteasomal activities has been recognized as a major contributor to the progression of cardiovascular diseases. Therefore, approaches to restore proteolytic function within the setting of the diseased myocardium would be of great clinical significance. In this study, we discovered that the cardiac proteasomal activity could be regulated by acetylation. Histone deacetylase (HDAC) inhibitors (suberoylanilide hydroxamic acid and sodium valproate) enhanced the acetylation of 20S proteasome subunits in the myocardium and led to an elevation of proteolytic capacity. This regulatory paradigm was present in both healthy and acutely ischemia/reperfusion (I/R) injured murine hearts, and HDAC inhibition in vitro restored proteolytic capacities to baseline sham levels in injured hearts. This mechanism of regulation was also viable in failing human myocardium. With 20S proteasomal complexes purified from murine myocardium treated with HDAC inhibitors in vivo, we confirmed that acetylation of 20S subunits directly, at least in part, presents a molecular explanation for the improvement in function. Furthermore, using high-resolution LC-MS/MS, we unraveled the first cardiac 20S acetylome, which identified the acetylation of nine N-termini and seven internal lysine residues. Acetylation on four lysine residues and four N-termini on cardiac proteasomes were novel discoveries of this study. In addition, the acetylation of five lysine residues was inducible via HDAC inhibition, which correlated with the enhancement of 20S proteasomal activity. Taken as a whole, our investigation unveiled a novel mechanism of proteasomal function regulation in vivo and established a new strategy for the potential rescue of compromised proteolytic function in the failing heart using HDAC inhibitors.

  8. Structure and Synaptic Function of Metal Binding to the Amyloid Precursor Protein and its Proteolytic Fragments

    PubMed Central

    Wild, Klemens; August, Alexander; Pietrzik, Claus U.; Kins, Stefan

    2017-01-01

    Alzheimer’s disease (AD) is ultimately linked to the amyloid precursor protein (APP). However, current research reveals an important synaptic function of APP and APP-like proteins (APLP1 and 2). In this context various neurotrophic and neuroprotective functions have been reported for the APP proteolytic fragments sAPPα, sAPPβ and the monomeric amyloid-beta peptide (Aβ). APP is a metalloprotein and binds copper and zinc ions. Synaptic activity correlates with a release of these ions into the synaptic cleft and dysregulation of their homeostasis is linked to different neurodegenerative diseases. Metal binding to APP or its fragments affects its structure and its proteolytic cleavage and therefore its physiological function at the synapse. Here, we summarize the current data supporting this hypothesis and provide a model of how these different mechanisms might be intertwined with each other. PMID:28197076

  9. Use of proteolytic enzymes as an additional tool for trypanosomatid identification.

    PubMed

    Santos, A L S; Abreu, C M; Alviano, C S; Soares, R M A

    2005-01-01

    The expression of proteolytic activities in the Trypanosomatidae family was explored as a potential marker to discriminate between the morphologically indistinguishable flagellates isolated from insects and plants. We have comparatively analysed the proteolytic profiles of 19 monoxenous trypanosomatids (Herpetomonas anglusteri, H. samuelpessoai, H. mariadeanei, H. roitmani, H. muscarum ingenoplastis, H. muscarum muscarum, H. megaseliae, H. dendoderi, Herpetomoas sp., Crithidia oncopelti, C. deanei, C. acanthocephali, C. harmosa, C. fasciculata, C. guilhermei, C. luciliae, Blastocrithidia culicis, Leptomonas samueli and Lept. seymouri) and 4 heteroxenous flagellates (Phytomonas serpens, P. mcgheei, Trypanosoma cruzi and Leishmania amazonensis) by in situ detection of enzyme activities on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE ) containing co-polymerized gelatine as substrate, in association with specific proteinase inhibitors. All 23 trypanosomatids expressed at least 1 acidic proteolytic enzyme. In addition, a characteristic and specific pattern of cell-associated metallo and/or cysteine proteinases was observed, except for the similar profiles detected in 2 Herpetomonas (H. anglusteri and H. samuelpessoai) and 3 Crithidia (C. fasciculata, C. guilhermei and C. luciliae) species. However, these flagellates released distinct secretory proteinase profiles into the extracellular medium. These findings strongly suggest that the association of cellular and secretory proteinase pattern could represent a useful marker to help trypanosomatid identification.

  10. The Caspase Proteolytic System in Callipyge and Normal Lambs in Longissimus, Semimembranosus, and Infraspinatus Muscles During Postmortem Storage

    USDA-ARS?s Scientific Manuscript database

    The objective of this experiment was to determine whether the caspase proteolytic system has a role in postmortem tenderization. Six ewes and six wethers that were non-carriers and six ewes and six wethers that were expressing the callipyge gene were used for this study. Caspase activities were de...

  11. The encephalomyocarditis virus 3C protease is a substrate for the ubiquitin-mediated proteolytic system.

    PubMed

    Lawson, T G; Gronros, D L; Werner, J A; Wey, A C; DiGeorge, A M; Lockhart, J L; Wilson, J W; Wintrode, P L

    1994-11-11

    The encephalomyocarditis virus 3C protease has been shown to be rapidly degraded in infected cells and in vitro in rabbit reticulocyte lysate. The in vitro degradation, at least, is accomplished by a virus-independent, ATP-dependent proteolytic system. Here we identify this proteolytic system as the ubiquitin-mediated system. Incubation of the 3C protease in rabbit reticulocyte or cultured mouse cell lysate preparations, alone or in the presence of added ubiquitin or methylated ubiquitin, resulted in the generation of new higher molecular weight species. These new products were shown to be 3C protease-ubiquitin conjugates by their ability to bind antibodies against both the 3C protease and ubiquitin. Supplemental ubiquitin also stimulated the degradation of the 3C protease in these preparations. Large 3C protease-polyubiquitin conjugates were observed to accumulate in reticulocyte lysate in the presence of adenosine 5'-O-(3-thiotriphosphate), an inhibitor of the 26 S multicatalytic protease. This, combined with the fact that the proteolytic activity could be removed from the lysate by sedimentation, implicates the multicatalytic protease in the degradation of the 3C protease-ubiquitin conjugates. It was also found that the slow rate of degradation of a model polyprotein, which resembles the stable viral 3CD diprotein produced in vivo, is likely due to the fact that the polyprotein is a poor substrate for the ubiquitin-conjugating system.

  12. Moderate and sustained exercise modulates muscle proteolytic and myogenic markers in gilthead sea bream (Sparus aurata).

    PubMed

    Vélez, Emilio J; Azizi, Sheida; Lutfi, Esmail; Capilla, Encarnación; Moya, Alberto; Navarro, Isabel; Fernández-Borràs, Jaume; Blasco, Josefina; Gutiérrez, Joaquim

    2017-05-01

    Swimming activity primarily accelerates growth in fish by increasing protein synthesis and energy efficiency. The role of muscle in this process is remarkable and especially important in teleosts, where muscle represents a high percentage of body weight and because many fish species present continuous growth. The aim of this work was to characterize the effects of 5 wk of moderate and sustained swimming in gene and protein expression of myogenic regulatory factors, proliferation markers, and proteolytic molecules in two muscle regions (anterior and caudal) of gilthead sea bream fingerlings. Western blot results showed an increase in the proliferation marker proliferating cell nuclear antigen (PCNA), proteolytic system members calpain 1 and cathepsin D, as well as vascular endothelial growth factor protein expression. Moreover, quantitative real-time PCR data showed that exercise increased the gene expression of proteases (calpains, cathepsins, and members of the ubiquitin-proteasome system in the anterior muscle region) and the gene expression of the proliferation marker PCNA and the myogenic factor MyoD in the caudal area compared with control fish. Overall, these data suggest a differential response of the two muscle regions during swimming adaptation, with tissue remodeling and new vessel formation occurring in the anterior muscle and enhanced cell proliferation and differentiation occurring in the caudal area. In summary, the present study contributes to improving the knowledge of the role of proteolytic molecules and other myogenic factors in the adaptation of muscle to moderate sustained swimming in gilthead sea bream. Copyright © 2017 the American Physiological Society.

  13. A Strong Neutrophil Elastase Proteolytic Fingerprint Marks the Carcinoma Tumor Proteome.

    PubMed

    Kistowski, Michał; Dębski, Janusz; Karczmarski, Jakub; Paziewska, Agnieszka; Olędzki, Jacek; Mikula, Michał; Ostrowski, Jerzy; Dadlez, Michał

    2017-02-01

    Proteolytic cascades are deeply involved in critical stages of cancer progression. During the course of peptide-wise analysis of shotgun proteomic data sets representative of colon adenocarcinoma (AC) and ulcerative colitis (UC), we detected a cancer-specific proteolytic fingerprint composed of a set of numerous protein fragments cleaved C-terminally to V, I, A, T, or C residues, significantly overrepresented in AC. A peptide set linked by a common VIATC cleavage consensus was the only prominent cancer-specific proteolytic fingerprint detected. This sequence consensus indicated neutrophil elastase as a source of the fingerprint. We also found that a large fraction of affected proteins are RNA processing proteins associated with the nuclear fraction and mostly cleaved within their functionally important RNA-binding domains. Thus, we detected a new class of cancer-specific peptides that are possible markers of tumor-infiltrating neutrophil activity, which often correlates with the clinical outcome. Data are available via ProteomeXchange with identifiers: PXD005274 (Data set 1) and PXD004249 (Data set 2). Our results indicate the value of peptide-wise analysis of large global proteomic analysis data sets as opposed to protein-wise analysis, in which outlier differential peptides are usually neglected.

  14. Proteolytic Processing of Interleukin-1 Family Cytokines: Variations on a Common Theme.

    PubMed

    Afonina, Inna S; Müller, Christina; Martin, Seamus J; Beyaert, Rudi

    2015-06-16

    Members of the extended interleukin-1 (IL-1) cytokine family, such as IL-1, IL-18, IL-33, and IL-36, play a pivotal role in the initiation and amplification of immune responses. However, deregulated production and/or activation of these cytokines can lead to the development of multiple inflammatory disorders. IL-1 family members share a broadly similar domain organization and receptor signaling pathways. Another striking similarity between IL-1 family members is the requirement for proteolytic processing in order to unlock their full biological potential. Although much emphasis has been put on the role of caspase-1, another emerging theme is the involvement of neutrophil- and mast cell-derived proteases in IL-1 family cytokine processing. Elucidating the regulation of IL-1 family members by proteolytic processing is of great interest for understanding inflammation and immunity. Here, we review the identity of the proteases involved in the proteolytic processing of IL-1 family cytokines and the therapeutic implications in inflammatory disease. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Zinc pyrithione inhibits caspase-3 activity, promotes ErbB1-ErbB2 heterodimerization and suppresses ErbB2 downregulation in cardiomyocytes subjected to ischemia/reperfusion.

    PubMed

    Bodiga, Vijaya Lakshmi; Thokala, Sandhya; Vemuri, Praveen Kumar; Bodiga, Sreedhar

    2015-12-01

    Heart tissue becomes zinc-depleted and the capacity to mobilize labile zinc is diminished, indicating zinc dyshomeostasis during ischemia/reperfusion (I/R). Apparently, zinc pyrithione restores the basal zinc levels during I/R and prevents apoptosis by activating phosphatidyl inositol-3-kinase/Akt and targeting mitochondrial permeability transition. Receptor tyrosine kinases of the ErbB family (ErbB1 to ErbB4) are cell surface proteins that can regulate cell growth, proliferation and survival. Previous studies have shown that zinc pyrithione-induced activation of PI3kinase/Akt requires ErbB2 expression. On the other hand, while I/R decreases ErbB2 levels causing cardiomyocyte dysfunction and cell death, zinc pyrithione restores ErbB2 levels and maintains cardiomyocyte function. H9c2 cells expressed all the four ErbBs, although the expression of ErbB1 and ErbB2 were higher compared to ErbB3 and ErbB4. Hypoxia/Reoxygenation (H/R) had opposing effects on the mRNA expression of ErbB1 and ErbB2. ErbB2 mRNA levels were enhanced, but corresponding ErbB2 protein levels decreased after reoxygenation. H/R induced the degradation of ErbB2 in caspase-3 dependent manner, with the formation of a 25kDa fragment. This fragment could be detected after H/R only upon treatment of the cells with a proteasomal inhibitor, ALLN, suggesting that caspase-mediated cleavage of 185kDa ErbB2 results in C-terminal cleavage and formation of 25kDa fragment, which is further degraded by proteasome. Heterodimerization and phosphorylation of ErbB2/ErbB1 which decreased upon reoxygenation, was promoted by zinc pyrithione. Zinc pyrithione effectively suppressed the caspase activation, decreased the proteolytic cleavage of ErbB2, enhanced the phosphorylation and activation of ErbB1-ErbB2 complexes and improved the cell survival after hypoxia/reoxygenation.

  16. Chemotherapy-induced mucositis is associated with changes in proteolytic pathways.

    PubMed

    Leblond, Jonathan; Le Pessot, Florence; Hubert-Buron, Aurélie; Duclos, Célia; Vuichoud, Jacques; Faure, Magali; Breuillé, Denis; Déchelotte, Pierre; Coëffier, Moïse

    2008-02-01

    Mucositis, a common toxic side effect of chemotherapy, is characterized by an arrest of cell proliferation and a loss of gut barrier function, which may cause treatment reduction or withdrawal. Gut integrity depends on nutritional and metabolic factors, including the balance between protein synthesis and proteolysis. The effects of methotrexate (MTX; a frequently used chemotherapeutic agent) on intestinal proteolysis and gut barrier function were investigated in rats. Male Sprague-Dawley rats received 2.5 mg/kg of MTX subcutaneously during 3 days and were euthanized at Day 4 (D4) or Day 7 (D7). We observed at D4 that MTX induced mucosal damage and increased intestinal permeability (7-fold) and the mucosal concentration of interleukin (IL)-1beta and IL-6 (4- to 6-fold). In addition, villus height and glutathione content significantly decreased. Intestinal proteolysis was also affected by MTX as cathepsin D activity increased at D4, whereas chymotrypsin-like proteasome activity decreased and calpain activities remained unaffected. At D7, cathepsin D activity was restored to control levels, but proteasome activity remained reduced. This disruption of proteolysis pathways strongly contributed to mucositis and requires further study. Lysosomal proteolytic activity may be considered the main proteolytic pathway responsible for alteration of mucosal integrity and intestinal permeability during mucositis, as cathepsin D activity was found to be correlated with mucosal atrophy and intestinal permeability. Proteasome regulation could possibly be an adaptive process for survival. Future investigation is warranted to target proteolytic pathways with protective nutritional or pharmacological therapies during mucositis.

  17. An ancestral non-proteolytic role for presenilin proteins in multicellular development of the social amoeba Dictyostelium discoideum.

    PubMed

    Ludtmann, Marthe H R; Otto, Grant P; Schilde, Christina; Chen, Zhi-Hui; Allan, Claire Y; Brace, Selina; Beesley, Philip W; Kimmel, Alan R; Fisher, Paul; Killick, Richard; Williams, Robin S B

    2014-04-01

    Mutations in either of two presenilin genes can cause familial Alzheimer's disease. Presenilins have both proteolysis-dependent functions, as components of the γ-secretase complex, and proteolysis-independent functions in signalling. In this study, we investigate a conserved function of human presenilins in the development of the simple model organism Dictyostelium discoideum. We show that the block in Dictyostelium development caused by the ablation of both Dictyostelium presenilins is rescued by the expression of human presenilin 1, restoring the terminal differentiation of multiple cell types. This developmental role is independent of proteolytic activity, because the mutation of both catalytic aspartates does not affect presenilin ability to rescue development, and the ablation of nicastrin, a γ-secretase component that is crucial for proteolytic activity, does not block development. The role of presenilins during Dictyostelium development is therefore independent of their proteolytic activity. However, presenilin loss in Dictyostelium results in elevated cyclic AMP (cAMP) levels and enhanced stimulation-induced calcium release, suggesting that presenilins regulate these intracellular signalling pathways. Our data suggest that presenilin proteins perform an ancient non-proteolytic role in regulating intracellular signalling and development, and that Dictyostelium is a useful model for analysing human presenilin function.

  18. Regulation of urokinase receptor proteolytic function by the tetraspanin CD82.

    PubMed

    Bass, Rosemary; Werner, Finn; Odintsova, Elena; Sugiura, Tsuyoshi; Berditchevski, Fedor; Ellis, Vincent

    2005-04-15

    The high affinity interaction between the urokinase-type plasminogen activator (uPA) and its glycolipid-anchored cellular receptor (uPAR) promotes plasminogen activation and the efficient generation of pericellular proteolytic activity. We demonstrate here that expression of the tetraspanin CD82/KAI1 (a tumor metastasis suppressor) leads to a profound effect on uPAR function. Pericellular plasminogen activation was reduced by approximately 50-fold in the presence of CD82, although levels of components of the plasminogen activation system were unchanged. uPAR was present on the cell surface and molecularly intact, but radioligand binding analysis with uPA and anti-uPAR antibodies revealed that it was in a previously undetected cryptic form unable to bind uPA. This was not due to direct interactions between uPAR and CD82, as they neither co-localized on the cell surface nor could be co-immunoprecipitated. However, expression of CD82 led to a redistribution of uPAR to focal adhesions, where it was shown by double immunofluorescence labeling to co-localize with the integrin alpha(5)beta(1), which was also redistributed in the presence of CD82. Co-immunoprecipitation experiments showed that, in the presence of CD82, uPAR preferentially formed stable associations with alpha(5)beta(1), but not with a variety of other integrins, including alpha(3)beta(1). These data suggest that CD82 inhibits the proteolytic function of uPAR indirectly, directing uPAR and alpha(5)beta(1) to focal adhesions and promoting their association with a resultant loss of uPA binding. This represents a novel mechanism whereby tetraspanins, integrins, and uPAR, systems involved in cell adhesion and migration, cooperate to regulate pericellular proteolytic activity and may suggest a mechanism for the tumor-suppressive effects of CD82/KAI1.

  19. A radiolabel-release microwell assay for proteolytic enzymes present in cell culture media

    SciTech Connect

    Rucklidge, G.J.; Milne, G. )

    1990-03-01

    A modified method for the measurement of proteolytic enzyme activity in cell culture-conditioned media has been developed. Using the release of 3H-labeled peptides from 3H-labeled gelatin the method is performed in microwell plates. The substrate is insolubilized and attached to the wells by glutaraldehyde treatment, thus eliminating the need for a precipitation step at the end of the assay. The assay is sensitive, reproducible, and convenient for small sample volumes. The effect of different protease inhibitors on activity can be assessed rapidly allowing an early characterization of the enzyme. It can also be adapted to microplate spectrophotometric analysis by staining residual substrate with Coomassie blue.

  20. Isolation of Microsporum gypseum in soil samples from different geographical regions of brazil, evaluation of the extracellular proteolytic enzymes activities (keratinase and elastase) and molecular sequencing of selected strains

    PubMed Central

    Giudice, Mauro Cintra; Reis-Menezes, Adriana Araújo; Rittner, Glauce Mary Gomes; Mota, Adolfo José; Gambale, Walderez

    2012-01-01

    A survey of Microsporum gypseum was conducted in soil samples in different geographical regions of Brazil. The isolation of dermatophyte from soil samples was performed by hair baiting technique and the species were identified by morphology studies. We analyzed 692 soil samples and the recuperating rate was 19.2%. The activities of keratinase and elastase were quantitatively performed in 138 samples. The sequencing of the ITS region of rDNA was performed in representatives samples. M. gypseum isolates showed significant quantitative differences in the expression of both keratinase and elastase, but no significant correlation was observed between these enzymes. The sequencing of the representative samples revealed the presence of two teleomorphic species of M. gypseum (Arthroderma gypseum and A. incurvatum). The enzymatic activities may play an important role in the pathogenicity and a probable adaptation of this fungus to the animal parasitism. Using the phenotypical and molecular analysis, the Microsporum identification and their teleomorphic states will provide a useful and reliable identification system. PMID:24031904

  1. A new mass-spectrometric C-terminal sequencing technique finds a similarity between gamma-interferon and alpha 2-interferon and identifies a proteolytically clipped gamma-interferon that retains full antiviral activity.

    PubMed Central

    Rose, K; Simona, M G; Offord, R E; Prior, C P; Otto, B; Thatcher, D R

    1983-01-01

    A novel mass-spectrometric technique is described that permits the identification of the C-terminal peptide of a protein. The technique involves the incorporation of 18O into all alpha-carboxy groups liberated during enzyme-catalysed partial hydrolysis of the protein, followed by mass spectrometry to identify as the C-terminal peptide the only peptide that did not incorporate any 18O. The technique has been used to identify the true C-terminal tryptic peptide of a bacterially produced gamma-interferon and to distinguish it from a peptide produced by anomalous tryptic cleavage. It was found that a closely similar sequence segment of bacterially produced alpha 2-interferon undergoes an analogous cleavage. The technique was also used to identify the C-terminus of a clipped gamma-interferon that retains full antiviral activity. PMID:6418141

  2. Design of potent, proteolytically stable oxyntomodulin analogs

    PubMed Central

    Muppidi, Avinash; Zou, Huafei; Yang, Peng Yu; Chao, Elizabeth; Sherwood, Lance; Nunez, Vanessa; Woods, Ashley

    2016-01-01

    Incretin-based peptides are effective therapeutics for treating type 2 diabetes mellitus (T2DM). Oxyntomodulin (OXM), a dual agonist of GLP-1R and GCGR, has shown superior weight loss and glucose lowering effects, compared to single GLP-1R agonists. To overcome the short half-life and rapid renal clearance of OXM, which limit its therapeutic potential, both lipid and PEG modified OXM analogs have been reported. However, these approaches often result in reduced potency or PEG-associated toxicity. Herein we report a new class of cross-linked OXM analogs that show increased plasma stability and higher potency in activating both GLP-1R and GCGR. Moreover, the extended in vivo half-life results in superior anti-hyperglycemic activity in mice compared to the wild-type OXM. PMID:26727558

  3. Proteolytic degradation of the RGD-binding and non-RGD-binding conformers of human platelet integrin glycoprotein IIb/IIIa: clues for identification of regions involved in the receptor's activation.

    PubMed Central

    Calvete, J J; Mann, K; Schäfer, W; Fernandez-Lafuente, R; Guisán, J M

    1994-01-01

    The human integrin glycoprotein (GP)IIb/IIIa plays a central role in haemostasis as an inducible receptor for fibrinogen and other RGD-containing adhesive proteins at the platelet plasma membrane. Expression of the fibrinogen receptor on platelet activation involves conformational changes in the quaternary structure of GPIIb/IIIa. Little is known, however, about the nature of this conformational transition. Given that isolated GPIIb/IIIa contains a mixture of RGD-binding and non-RGD-binding heterodimers, we used limited proteolysis as a tool for investigating the structural differences between the two conformers. Comparison of their fragmentation patterns shows that, whereas in the non-RGD-binding form of GPIIb/IIIa the N-terminal half of the heavy chain of GPIIb (GPIIbH) and the central region of GPIIIa are cleaved by endoproteinase Arg-C, these domains associate tightly with one another in the RGD-binding GPIIb/IIIa and are thus protected from proteolysis. In addition, the C-terminal half of GPIIb becomes more susceptible to degradation in the non-RGD-binding GPIIb/IIIa conformer. Our interpretation, in the context of available structural and functional data, is that a major relative reorientation of the GPIIbH and GPIIIa extracellular domains takes place along the subunit interface during the conformational transition of the platelet integrin. Images Figure 1 PMID:8129707

  4. Plastidial Starch Phosphorylase in Sweet Potato Roots Is Proteolytically Modified by Protein-Protein Interaction with the 20S Proteasome

    PubMed Central

    Lin, Yi-Chen; Chen, Han-Min; Chou, I-Min; Chen, An-Na; Chen, Chia-Pei; Young, Guang-Huar; Lin, Chi-Tsai; Cheng, Chiung-Hsiang; Chang, Shih-Chung; Juang, Rong-Huay

    2012-01-01

    Post-translational regulation plays an important role in cellular metabolism. Earlier studies showed that the activity of plastidial starch phosphorylase (Pho1) may be regulated by proteolytic modification. During the purification of Pho1 from sweet potato roots, we observed an unknown high molecular weight complex (HX) showing Pho1 activity. The two-dimensional gel electrophoresis, mass spectrometry, and reverse immunoprecipitation analyses showed that HX is composed of Pho1 and the 20S proteasome. Incubating sweet potato roots at 45°C triggers a stepwise degradation of Pho1; however, the degradation process can be partially inhibited by specific proteasome inhibitor MG132. The proteolytically modified Pho1 displays a lower binding affinity toward glucose 1-phosphate and a reduced starch-synthesizing activity. This study suggests that the 20S proteasome interacts with Pho1 and is involved in the regulation of the catalytic activity of Pho1 in sweet potato roots under heat stress conditions. PMID:22506077

  5. Plastidial starch phosphorylase in sweet potato roots is proteolytically modified by protein-protein interaction with the 20S proteasome.

    PubMed

    Lin, Yi-Chen; Chen, Han-Min; Chou, I-Min; Chen, An-Na; Chen, Chia-Pei; Young, Guang-Huar; Lin, Chi-Tsai; Cheng, Chiung-Hsiang; Chang, Shih-Chung; Juang, Rong-Huay

    2012-01-01

    Post-translational regulation plays an important role in cellular metabolism. Earlier studies showed that the activity of plastidial starch phosphorylase (Pho1) may be regulated by proteolytic modification. During the purification of Pho1 from sweet potato roots, we observed an unknown high molecular weight complex (HX) showing Pho1 activity. The two-dimensional gel electrophoresis, mass spectrometry, and reverse immunoprecipitation analyses showed that HX is composed of Pho1 and the 20S proteasome. Incubating sweet potato roots at 45°C triggers a stepwise degradation of Pho1; however, the degradation process can be partially inhibited by specific proteasome inhibitor MG132. The proteolytically modified Pho1 displays a lower binding affinity toward glucose 1-phosphate and a reduced starch-synthesizing activity. This study suggests that the 20S proteasome interacts with Pho1 and is involved in the regulation of the catalytic activity of Pho1 in sweet potato roots under heat stress conditions.

  6. Crystal structures, spectroscopic features, and catalytic properties of cobalt(II), copper(II), nickel(II), and mercury(II) derivatives of the zinc endopeptidase astacin. A correlation of structure and proteolytic activity.

    PubMed

    Gomis-Rüth, F X; Grams, F; Yiallouros, I; Nar, H; Küsthardt, U; Zwilling, R; Bode, W; Stöcker, W

    1994-06-24

    The catalytic zinc ion of astacin, a prototypical metalloproteinase from crayfish, has been substituted by Co(II), Cu(II), Hg(II), and Ni(II) in order to probe the role of the metal for both catalysis and structure. Compared to Zn(II)-astacin, Co(II)- and Cu(II)-astacin display enzymatic activities of about 140 and 37%, respectively, while Ni(II)- and Hg(II)-astacin are almost inactive. The electron paramagnetic resonance spectrum of Cu(II)-astacin is typical of 5-fold coordinated copper(II), and its intense absorption maxima at 445 and 325 nm are probably due to ligand-metal charge-transfer transitions involving Tyr-149. This residue had been identified previously by x-ray crystallography of the zinc enzyme as a zinc ligand, in addition to three imidazoles and a glutamic acid-bound water molecule. We present now the refined high-resolution x-ray crystal structures of Cu(II)-, Co(II)-, and Ni(II)-astacin, which exhibit a virtually identical protein framework to the previously analyzed structures of Zn(II)-, apo-, and Hg(II)-astacin. In Co(II)- and Cu(II)-astacin, the metal is penta-coordinated similarly to the native zinc enzyme. In the Ni(II) derivative, however, an additional solvent molecule expands the metal coordination sphere to a distorted octahedral ligand geometry, while in Hg(II)-astacin, no ordered solvent molecule at all is observed in the inner coordination sphere of the metal. This indicates a close correlation between catalytic properties and ground-state metal coordination of astacin.

  7. Complex Negative Regulation of TLR9 by Multiple Proteolytic Cleavage Events.

    PubMed

    Sinha, Siddhartha S; Cameron, Jody; Brooks, James C; Leifer, Cynthia A

    2016-08-15

    TLR9 is an innate immune receptor important for recognizing DNA of host and foreign origin. A mechanism proposed to prevent excessive response to host DNA is the requirement for proteolytic cleavage of TLR9 in endosomes to generate a mature form of the receptor (TLR9(471-1032)). We previously described another cleavage event in the juxtamembrane region of the ectodomain that generated a dominant-negative form of TLR9. Thus, there are at least two independent cleavage events that regulate TLR9. In this study, we investigated whether an N-terminal fragment of TLR9 could be responsible for regulation of the mature or negative-regulatory form. We show that TLR9(471-1032), corresponding to the proteolytically cleaved form, does not function on its own. Furthermore, activity is not rescued by coexpression of the N-terminal fragment (TLR9(1-440)), inclusion of the hinge region (TLR9(441-1032)), or overexpression of UNC93B1, the last of which is critical for trafficking and cleavage of TLR9. TLR9(1-440) coimmunoprecipitates with full-length TLR9 and TLR9(471-1032) but does not rescue the native glycosylation pattern; thus, inappropriate trafficking likely explains why TLR9(471-1032) is nonfunctional. Lastly, we show that TLR9(471-1032) is also a dominant-negative regulator of TLR9 signaling. Together, these data provide a new perspective on the complexity of TLR9 regulation by proteolytic cleavage and offer potential ways to inhibit activity through this receptor, which may dampen autoimmune inflammation.

  8. Partial Characterization of the Proteolytic Properties of an Enzymatic Extract From "Aguama" Bromelia pinguin L. Fruit Grown in Mexico.

    PubMed

    Moreno-Hernández, Jesús Martín; Hernández-Mancillas, Xitlalli Desideria; Navarrete, Evelia Lorena Coss; Mazorra-Manzano, Miguel Ángel; Osuna-Ruiz, Idalia; Rodríguez-Tirado, Víctor Alfonso; Salazar-Leyva, Jesús Aarón

    2017-05-01

    Plant proteases are capable of performing several functions in biological systems, and their use is attractive for biotechnological process due to their interesting catalytic properties. Bromelia pinguin (aguama) is a wild abundant natural resource in several regions of Central America and the Caribbean Islands but is underutilized. Their fruits are rich in proteases with properties that are still unknown, but they represent an attractive source of enzymes for biotechnological applications. Thus, the proteolytic activity in enzymatic crude extracts (CEs) from wild B. pinguin fruits was partially characterized. Enzymes in CEs showed high proteolytic activity at acid (pH 2.0-4.0) and neutral alkaline (pH 7.0-9.0) conditions, indicating that different types of active proteases are present. Proteolytic activity inhibition by the use of specific protease inhibitors indicated that aspartic, cysteine, and serine proteases are the main types of proteases present in CEs. Activity at pH 3.0 was stable in a broad range of temperatures (25-50 °C) and retained its activity in the presence of surfactants (SDS, Tween-80), reducing agents (DTT, 2-mercapoethanol), and organic solvents (methanol, ethanol, acetone, 2-propanol), which suggests that B. pinguin proteases are potential candidates for their application in brewing, detergent, and pharmaceutical industries.

  9. Non-proteolytic functions of microbial proteases increase pathological complexity.

    PubMed

    Jarocki, Veronica M; Tacchi, Jessica L; Djordjevic, Steven P

    2015-03-01

    Proteases are enzymes that catalyse hydrolysis of peptide bonds thereby controlling the shape, size, function, composition, turnover and degradation of other proteins. In microbes, proteases are often identified as important virulence factors and as such have been targets for novel drug design. It is emerging that some proteases possess additional non-proteolytic functions that play important roles in host epithelia adhesion, tissue invasion and in modulating immune responses. These additional "moonlighting" functions have the potential to obfuscate data interpretation and have implications for therapeutic design. Moonlighting enzymes comprise a subcategory of multifunctional proteins that possess at least two distinct biological functions on a single polypeptide chain. Presently, identifying moonlighting proteins relies heavily on serendipitous empirical data with clues arising from proteins lacking signal peptides that are localised to the cell surface. Here, we describe examples of microbial proteases with additional non-proteolytic functions, including streptococcal pyrogenic exotoxin B, PepO and C5a peptidases, mycoplasmal aminopeptidases, mycobacterial chaperones and viral papain-like proteases. We explore how these non-proteolytic functions contribute to host cell adhesion, modulate the coagulation pathway, assist in non-covalent folding of proteins, participate in cell signalling, and increase substrate repertoire. We conclude by describing how proteomics has aided in moonlighting protein discovery, focusing attention on potential moonlighters in microbial exoproteomes.

  10. The role of APP proteolytic processing in lipid metabolism.

    PubMed

    Grimm, Marcus O W; Rothhaar, Tatjana L; Hartmann, Tobias

    2012-04-01

    Amyloid plaques in brains are one of the major pathological hallmarks of Alzheimer's disease (AD). These plaques are mainly formed by aggregated Aβ, generated by proteolytic cleavage of the amyloid precursor protein (APP). Therefore, APP processing and Aβ production have been one of the central scopes in AD research in the past. Now, accumulating evidence suggests that besides its pathological impact, APP and its cleavage products also contribute to physiological functions. Proteolytic cleavage of APP is tightly regulated, and several lipids such as cholesterol and sphingolipids have been shown to influence APP processing and Aβ generation. In turn, Aβ as well as other APP cleavage products plays an essential role in regulating lipid homeostasis arguing for complex regulatory cycles in which lipids control APP processing and vice versa. This balanced regulation is disrupted under pathological conditions such as in AD. This article will review the physiological function of APP and its proteolytic products, especially Aβ and AICD, in regulating lipid homeostasis and which lipid species modulate APP processing. Furthermore, we summarize the alterations in lipid metabolism observed in AD patients and AD mouse models.

  11. Bicarbonate-Dependent Secretion and Proteolytic Processing of Recombinant Myocilin

    PubMed Central

    Aroca-Aguilar, José-Daniel; Martínez-Redondo, Francisco; Martín-Gil, Alba; Pintor, Jesús; Coca-Prados, Miguel; Escribano, Julio

    2013-01-01

    Myocilin is an extracellular glycoprotein of poorly understood function. Mutations of this protein are involved in glaucoma, an optic neuropathy characterized by a progressive and irreversible visual loss and frequently associated with elevated intraocular pressure. We previously showed that recombinant myocilin undergoes an intracellular proteolytic processing by calpain II which cleaves the central region of the protein, releasing one N- and one C-terminal fragment. Myocilin cleavage is reduced by glaucoma mutations and it has been proposed to participate in intraocular pressure modulation. To identify possible factors regulating the proteolytic processing of recombinant myocilin, we used a cellular model in which we analyzed how different culture medium parameters (i.e., culture time, cell density, pH, bicarbonate concentration, etc.) affect the presence of the extracellular C-terminal fragment. Extracellular bicarbonate depletion associated with culture medium acidification produced a reversible intracellular accumulation of full-length recombinant myocilin and incremented its intracellular proteolytic processing, raising the extracellular C-terminal fragment percentage. It was also determined that myocilin intracellular accumulation depends on its N-terminal region. These data suggest that aqueous humor bicarbonate variations could also modulate the secretion and cleavage of myocilin present in ocular tissues. PMID:23342144

  12. Does inhibition of proteolytic activity improve adhesive luting?

    PubMed

    Lührs, Anne-Katrin; De Munck, Jan; Geurtsen, Werner; Van Meerbeek, Bart

    2013-04-01

    Endogenous enzymes may be involved in the biodegradation of adhesive restoration-tooth interfaces. Inhibitors of matrix metalloproteinases (MMPs) have been suggested to retard the bond-degradation process. Limited data are available on whether composite cements may also benefit from MMP inhibitors. Therefore, the aim of this study was to determine the effect of two MMP inhibitors--chlorhexidine digluconate (CHX) and galardin--on the microtensile bond strength (μTBS) of two self-adhesive composite cements to dentin. Ceramic specimens were cemented to bur-cut dentin surfaces using the self-adhesive composite cements RelyX Unicem 2 (3M ESPE) or Clearfil SA (Kuraray), or the etch-and-rinse composite cement Nexus 3 (Kerr) that served as the control. The surfaces were left untreated or were pretreated with MMP inhibitors (2% CHX or 0.2 mM galardin). The μTBS was determined 'immediately' and upon ageing (water storage for 6 months). Statistical analysis revealed a significant effect of the factors 'composite cement' and 'storage', as well as all interactions, but no effect of the MMP inhibitors. After 6 months of ageing, the μTBS decreased for all cements, except for the multistep etch-and-rinse luting composite when it was applied without MMP inhibitors. The MMP inhibitors could not prevent the decrease in μTBS upon ageing and therefore do not improve the luting durability of the composite cements tested.

  13. Production of proteolytic enzymes in mast cells, fibroblasts, vascular smooth muscle and endothelial cells cultivated under normoxic or hypoxic conditions.

    PubMed

    Maxová, H; Bačáková, L; Lisá, V; Novotná, J; Tomášová, H; Vízek, M; Herget, J

    2010-01-01

    Matrix metalloproteinases (MMPs) is a family of proteolytic enzymes involved in remodeling of extracellular matrix. Although proteolytic enzymes are produced by many cell types, mast cells seem to be more important than other types in remodeling of pulmonary arteries during hypoxia. Therefore, we tested in vitro production of MMPs and serine proteases in four cell types (mast cells, fibroblasts, vascular smooth muscle cells and endothelial cells) cultivated for 48 h under normoxic or hypoxic (3% O2) conditions. MMP-13 was visualized by immunohistochemistry, MMP-2 and MMP-9 were detected by zymography in cell lysates. Enzymatic activities (MMPs, tryptase and chymase) were estimated in the cultivation media. Hypoxia had a minimal effect on total MMP activity in the cultivation media of all types of cells, but immunofluorescence revealed higher intensity of MMP-13 in the cells exposed to hypoxia except of fibroblasts. Tryptase activity was three times higher and chymase activity twice higher in mast cells cultivated in hypoxia than in those cultured in normoxia. Among all cell types studied here, mast cells are the most abundant source of proteolytic enzymes under normoxic and hypoxic conditions. Moreover, in these cells hypoxia increases the production of both specific serine proteases tryptase and chymase, which can act as MMPs activators.

  14. Peptide bond cleavage site determination of novel proteolytic enzymes found in ROS 17/2.8 cell lysates.

    PubMed

    Guidon, P T; Perrin, D; Harrison, P

    1996-02-01

    We have identified proteolytic activities in the rat osteoblastic osteosarcoma cell line ROS 17/2.8 which are capable of cleaving a peptide substrate for protein kinase C-mediated phosphorylation (PSPKC, Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys). Using polyacrylamide gel electrophoresis conditions similar to those used to resolve small molecular weight proteins, the peptide bonds of PSPKC which are cleaved by the proteolytic activities present in ROS 17/2.8 cell lysates have been determined. These activities cleave the Ser-Arg, Thr-Leu, and Ser-Val peptide bonds. To date, no proteolytic activities present in osteoblast cell lysates have been described with the aforementioned peptide bond specificities, suggesting that these activities are novel. The PSPKC-cleaved peptide fragment pattern generated was similar for several different osteoblast cell lysates. Lysates generated from different rat tissues were also able to cleave PSPKC, but the peptide fragment pattern generated by ROS 17/2.8 cell lysates appeared to be unique amongst these tissues.

  15. The polyphenol-rich extract from grape seeds inhibits platelet signaling pathways triggered by both proteolytic and non-proteolytic agonists.

    PubMed

    Olas, Beata; Wachowicz, Barbara; Stochmal, Anna; Oleszek, Wiesław

    2012-01-01

    Mechanisms involved in the reduction of blood platelet functions by various plant extract, including the grape seeds extract (rich in phenolic compounds, a mixture of about 95% oligomeric phenols; GSE) are still unclear. In the literature there are few papers describing studies on the effects of GSE on selected element of hemostasis. The aim of our study was to establish and compare the influence of GSE (at final dose of 0.625-50 µg/ml) and resveratrol (3,4',5 - trihydroxystilben), a phenolic compound synthesized in grapes and vegetables and presents in wine, which has been supposed to be beneficial for the prevention of cardiovascular events, on different steps of platelet activation. We measured the effects of GSE and resveratrol on platelet aggregation, the surface expression of P-selectin, platelet microparticle formation (PMP), and superoxide anion radicals ([Formula: see text]) production in blood platelets stimulated by TRAP and thrombin. P-selectin expression and PMP formation were measured by a flow cytometer. In gel-filtered platelets activated by thrombin or TRAP and treated with different concentrations of GSE (1.25-50 µg/ml) a significant decrease of P-selectin expression, PMP formation and platelet aggregation was observed. GSE caused also a dose-dependent reduction of [Formula: see text] produced in platelets activated by TRAP or thrombin. Our present results indicate that GSE inhibits platelet signaling pathways trigged by both proteolytic (thrombin) and non-proteolytic agonist (TRAP). In the comparative studies, GSE was found to be more effective antiplatelet factor, than the solution of pure resveratrol. Thus, the polyphenol-rich extract from grape seeds can be useful as the protecting factor against cardiovascular diseases.

  16. Non-proteolytic functions of calpain-3 in sarcoplasmic reticulum in skeletal muscles.

    PubMed

    Ojima, Koichi; Ono, Yasuko; Ottenheijm, Coen; Hata, Shoji; Suzuki, Hidenori; Granzier, Henk; Sorimachi, Hiroyuki

    2011-04-01

    Mutations in CAPN3/Capn3, which codes for skeletal muscle-specific calpain-3/p94 protease, are responsible for limb-girdle muscular dystrophy type 2A. Using "knock-in" (referred to as Capn3(CS/CS)) mice, in which the endogenous calpain-3 is replaced with a mutant calpain-3:C129S, which is a proteolytically inactive but structurally intact calpain-3, we demonstrated in our previous studies that loss of calpain-3 protease activity causes muscular dystrophy [Ojima, K. et al. (2010) J. Clin. Invest. 120, 2672-2683]. However, compared to Capn3-null (Capn3(-/-)) mice, Capn3(CS/CS) mice showed less severe dystrophic symptoms. This suggests that calpain-3 also has a non-proteolytic function. This study aimed to elucidate the non-proteolytic functions of calpain-3 through comparison of Capn3(CS/CS) mice with Capn3(-/-) mice. We found that calpain-3 is a component of the sarcoplasmic reticulum (SR), and that calpain-3 interacts with, but does not proteolyze, typical SR components such as ryanodine receptor and calsequestrin. Furthermore, Capn3(CS/CS) mice showed that the nonenzymatic role of calpain-3 is required for proper Ca(2+) efflux from the SR to cytosol during muscle contraction. These results indicate that calpain-3 functions as a nonenzymatic element for the Ca(2+) efflux machinery in the SR, rather than as a protease. Thus, defects in the nonenzymatic function of calpain-3 must also be involved in the pathogenesis of limb-girdle muscular dystrophy type 2A.

  17. Low-resolution structure of the proteolytic fragments of the Rapana venosa hemocyanin in solution.

    PubMed

    Dainese, E; Svergun, D; Beltramini, M; Di Muro, P; Salvato, B

    2000-01-01

    Rapana venosa hemocyanin (Hc) is a giant oxygen-binding protein consisting of different subunits assembled in a hollow cylinder. The polypeptide chain of each subunit is believed to be folded in several oxygen-binding functional units of molecular mass 50 kDa, each containing a binuclear copper active site. Limited proteolysis with alpha-chymotrypsin of native R. venosa hemocyanin allows the separation of three functional proteolytic fragments with molecular masses of approximately 150, 100, and 50 kDa. The functional fragments, purified by combining gel filtration chromatography and ion-exchange FPLC, were analyzed by means of small-angle X-ray scattering (SAXS). The gyration radius of the 50-kDa Rapana Hc fraction (2.4 nm) agrees well with that calculated on the basis of the dimensions determined by X-ray crystallography for one functional unit of Octopus Hc (2.1 nm). Independent shape determination of the 50- and 100-kDa proteolytic fragments yields consistent low-resolution models. Simultaneous fitting of the SAXS data from these fragments provides a higher-resolution model of the 100-kDa species made of two functional units tilted with respect to each other. The model of the 150-kDa proteolytic fragment consistent with the SAXS data displays a linear chain-like aggregation of the 50-kDa functional units. These observations provide valuable information for the reconstruction of the three-dimensional structure of the minimal functional subunit of gastropod hemocyanin in solution. Furthermore, the spatial relationships among the different functional units within the subunit will help in elucidation of the overall quaternary structure of the oligomeric native protein. Copyright 2000 Academic Press.

  18. A mature and fusogenic form of the Nipah virus fusion protein requires proteolytic processing by cathepsin L

    SciTech Connect

    Pager, Cara Theresia; Craft, Willie Warren; Patch, Jared; Dutch, Rebecca Ellis . E-mail: rdutc2@uky.edu

    2006-03-15

    The Nipah virus fusion (F) protein is proteolytically processed to F{sub 1} + F{sub 2} subunits. We demonstrate here that cathepsin L is involved in this important maturation event. Cathepsin inhibitors ablated cleavage of Nipah F. Proteolytic processing of Nipah F and fusion activity was dramatically reduced in cathepsin L shRNA-expressing Vero cells. Additionally, Nipah virus F-mediated fusion was inhibited in cathepsin L-deficient cells, but coexpression of cathepsin L restored fusion activity. Both purified cathepsin L and B could cleave immunopurified Nipah F protein, but only cathepsin L produced products of the correct size. Our results suggest that endosomal cathepsins can cleave Nipah F, but that cathepsin L specifically converts Nipah F to a mature and fusogenic form.

  19. Suppression of Aβ toxicity by puromycin-sensitive aminopeptidase is independent of its proteolytic activity☆

    PubMed Central

    Kruppa, Antonina J.; Ott, Stanislav; Chandraratna, Dhia S.; Irving, James A.; Page, Richard M.; Speretta, Elena; Seto, Tiffany; Camargo, Luiz Miguel; Marciniak, Stefan J.; Lomas, David A.; Crowther, Damian C.

    2013-01-01

    The accumulation of β-amyloid (Aβ) peptide in the brain is one of the pathological hallmarks of Alzheimer's disease and is thought to be of primary aetiological significance. In an unbiased genetic screen, we identified puromycin-sensitive aminopeptidase (PSA) as a potent suppressor of Aβ toxicity in a Drosophila model system. We established that coexpression of Drosophila PSA (dPSA) in the flies' brains improved their lifespan, protected against locomotor deficits, and reduced brain Aβ levels by clearing the Aβ plaque-like deposits. However, confocal microscopy and subcellular fractionation of amyloid-expressing 7PA2 cells demonstrated that PSA localizes to the cytoplasm. Therefore, PSA and Aβ are unlikely to be in the same cellular compartment; moreover, when we artificially placed them in the same compartment in flies, we could not detect a direct epistatic interaction. The consequent hypothesis that PSA's suppression of Aβ toxicity is indirect was supported by the finding that Aβ is not a proteolytic substrate for PSA in vitro. Furthermore, we showed that the enzymatic activity of PSA is not required for rescuing Aβ toxicity in neuronal SH-SY5Y cells. We investigated whether the stimulation of autophagy by PSA was responsible for these protective effects. However PSA's promotion of autophagosome fusion with lysosomes required proteolytic activity and so its effect on autophagy is not identical to its protection against Aβ toxicity. PMID:23911349

  20. Cell-penetrating TAT peptide in drug delivery systems: Proteolytic stability requirements

    PubMed Central

    Koren, Erez; Apte, Anjali; Sawant, Rupa R.; Grunwald, Jacob; Torchilin, Vladimir P.

    2012-01-01

    The stability and activity of the HIV cell-penetrating TAT peptide (TATp) on the surface of TATp-modified micelles and liposomes in relation to its proteolytic cleavage was investigated. TATp moieties were attached to the surface of these nanocarriers using TATp modified with a conjugate of phosphatidyl ethanolamine with a ‘short’ PEG (PEG-PE). Following pre-incubation with trypsin, elastase, or collagenase, the proteolytic stability of TATp on the surface of these modified carriers was studied by HPLC with fluorescence detection using fluorenylmethyl chloroformate (FMOC) labeling. All tested enzymes produced a dose-dependent cleavage of TATp as shown by the presence of TATp Arg-Arg fragments. Inhibition of TATp cleavage occurred when these TATp-micelles were modified by the addition of longer PEG-PE blocks, indicating an effective shielding of TATp from proteolysis by these blocks. TATp-modified carriers were also tested for their ability to accumulate in EL-4, HeLa, and B16-F10 cells. Trypsin treatment of TATp-modified liposomes and micelles resulted in decreased uptake and cell interaction, as measured by fluorescence microscopy and fluorescence-activated cell sorting techniques. Furthermore, a decrease in the cytotoxicity of TATp-modified liposomes loaded with doxorubicin (Doxil) was observed following trypsin treatment. In conclusion, steric shielding of TATp is essential to ensure its in vivo therapeutic function. PMID:21438724

  1. An evaluation of the proteolytic and lipolytic potential of Penicillium spp. isolated from traditional Greek sausages in submerged fermentation.

    PubMed

    Papagianni, Maria

    2014-01-01

    A number of novel Penicillium strains belonging to Penicillium nalgiovense, Penicillium solitum, Penicillium commune, Penicillium olsonii, and Penicillium oxalicum species, isolated from the surface of traditional Greek sausages, were evaluated for their proteolytic and lipolytic potential in a solid substrate first and next in submerged fermentations, using complex media. Extracellular proteolytic activity was assessed at acid, neutral, and alkaline pH, while the lipolytic activity was assessed using olive oil, the short-chain triacylglycerol tributyrin, and the long-chain triolein, as substrates. The study revealed that although closely related, the tested strains produce enzymes of distinct specificities. P. nalgiovense PNA9 produced the highest alkaline proteolytic activity (13.2 unit (U)/ml) and the highest lipolytic activity with tributyrin (92 U/ml). Comparisons with known sources show that proteases and/or lipases can be secreted effectively by some Penicillia (P. nalgiovense PNA4, PNA7, and PNA9 and P. solitum PSO1), and further investigations on their properties and characteristics would be promising.

  2. Influence of Amitraz and Oxalic Acid on the Cuticle Proteolytic System of Apis mellifera L. Workers

    PubMed Central

    Strachecka, Aneta; Paleolog, Jerzy; Olszewski, Krzysztof; Borsuk, Grzegorz

    2012-01-01

    This work verifies that amitraz and oxalic acid treatment affect honeybee cuticle proteolytic enzymes (CPE). Three bee groups were monitored: oxalic acid treatment, amitraz treatment, control. Electrophoresis of hydrophilic and hydrophobic CPE was performed. Protease and protease inhibitor activities (in vitro) and antifungal/antibacterial efficiencies (in vivo), were analyzed. Amitraz and oxalic acid treatment reduced hydrophobic, but did not affect hydrophilic, protein concentrations and reduced both hydrophilic and hydrophobic body surface asparagine and serine protease activities in relation to most substrates and independently of pH. The activities of natural cuticle inhibitors of acidic, neutral, and alkaline proteases were suppressed as a result of the treatments, corresponding with reduced antifungal and antibacterial activity. Electrophoretic patterns of low-, medium-, and high-molecular-weight proteases and protease inhibitors were also affected by the treatments. PMID:26466630

  3. Influence of Amitraz and Oxalic Acid on the Cuticle Proteolytic System of Apis mellifera L. Workers.

    PubMed

    Strachecka, Aneta; Paleolog, Jerzy; Olszewski, Krzysztof; Borsuk, Grzegorz

    2012-08-27

    This work verifies that amitraz and oxalic acid treatment affect honeybee cuticle proteolytic enzymes (CPE). Three bee groups were monitored: oxalic acid treatment, amitraz treatment, control. Electrophoresis of hydrophilic and hydrophobic CPE was performed. Protease and protease inhibitor activities (in vitro) and antifungal/antibacterial efficiencies (in vivo), were analyzed. Amitraz and oxalic acid treatment reduced hydrophobic, but did not affect hydrophilic, protein concentrations and reduced both hydrophilic and hydrophobic body surface asparagine and serine protease activities in relation to most substrates and independently of pH. The activities of natural cuticle inhibitors of acidic, neutral, and alkaline proteases were suppressed as a result of the treatments, corresponding with reduced antifungal and antibacterial activity. Electrophoretic patterns of low-, medium-, and high-molecular-weight proteases and protease inhibitors were also affected by the treatments.

  4. Quercetin induces apoptosis by activating caspase-3 and regulating Bcl-2 and cyclooxygenase-2 pathways in human HL-60 cells.

    PubMed

    Niu, Guomin; Yin, Songmei; Xie, Shuangfeng; Li, Yiqing; Nie, Danian; Ma, Liping; Wang, Xiuju; Wu, Yudan

    2011-01-01

    Quercetin is one of the naturally occurring dietary flavonol compounds. It is present abundantly in plants and has chemopreventive and anticancer effects. To investigate its anticancer mechanism, we examined the activity of quercetin against acute leukemia cell line, HL-60. Our results showed that quercetin inhibited cell proliferation and induced apoptosis in a time- and dose-dependent manner. Furthermore, quercetin down-regulated the expression of anti-apoptosis protein Bcl-2 and up-regulated the expression of pro-apoptosis protein Bax. Caspase-3 was also activated by quercetin, which started a caspase-3-depended mitochodrial pathway to induce apoptosis. It was also found that quercetin inhibited the expression of the cycloocygenase-2 (Cox-2) mRNA and Cox-2 protein. Taken together, these findings suggested that quercetin induces apoptosis in a caspase-3-dependent pathway by inhibiting Cox-2 expression and regulates the expression of downstream apoptotic components, including Bcl-2 and Bax. Quercetin can be a potent and promising medicine which might be safely used in leukemia therapy.

  5. The Xanthomonas campestris type III effector XopJ proteolytically degrades proteasome subunit RPT6.

    PubMed

    Üstün, Suayib; Börnke, Frederik

    2015-05-01

    Many animal and plant pathogenic bacteria inject type III effector (T3E) proteins into their eukaryotic host cells to suppress immunity. The Yersinia outer protein J (YopJ) family of T3Es is a widely distributed family of effector proteins found in both animal and plant pathogens, and its members are highly diversified in virulence functions. Some members have been shown to possess acetyltransferase activity; however, whether this is a general feature of YopJ family T3Es is currently unknown. The T3E Xanthomonas outer protein J (XopJ), a YopJ family effector from the plant pathogen Xanthomonas campestris pv vesicatoria, interacts with the proteasomal subunit Regulatory Particle AAA-ATPase6 (RPT6) in planta to suppress proteasome activity, resulting in the inhibition of salicylic acid-related immune responses. Here, we show that XopJ has protease activity to specifically degrade RPT6, leading to reduced proteasome activity in the cytoplasm as well as in the nucleus. Proteolytic degradation of RPT6 was dependent on the localization of XopJ to the plasma membrane as well as on its catalytic triad. Mutation of the Walker B motif of RPT6 prevented XopJ-mediated degradation of the protein but not XopJ interaction. This indicates that the interaction of RPT6 with XopJ is dependent on the ATP-binding activity of RPT6, but proteolytic cleavage additionally requires its ATPase activity. Inhibition of the proteasome impairs the proteasomal turnover of Nonexpressor of Pathogenesis-Related1 (NPR1), the master regulator of salicylic acid responses, leading to the accumulation of ubiquitinated NPR1, which likely interferes with the full induction of NPR1 target genes. Our results show that YopJ family T3Es are not only highly diversified in virulence function but also appear to possess different biochemical activities. © 2015 American Society of Plant Biologists. All Rights Reserved.

  6. The Xanthomonas campestris Type III Effector XopJ Proteolytically Degrades Proteasome Subunit RPT61[OPEN

    PubMed Central

    2015-01-01

    Many animal and plant pathogenic bacteria inject type III effector (T3E) proteins into their eukaryotic host cells to suppress immunity. The Yersinia outer protein J (YopJ) family of T3Es is a widely distributed family of effector proteins found in both animal and plant pathogens, and its members are highly diversified in virulence functions. Some members have been shown to possess acetyltransferase activity; however, whether this is a general feature of YopJ family T3Es is currently unknown. The T3E Xanthomonas outer protein J (XopJ), a YopJ family effector from the plant pathogen Xanthomonas campestris pv vesicatoria, interacts with the proteasomal subunit Regulatory Particle AAA-ATPase6 (RPT6) in planta to suppress proteasome activity, resulting in the inhibition of salicylic acid-related immune responses. Here, we show that XopJ has protease activity to specifically degrade RPT6, leading to reduced proteasome activity in the cytoplasm as well as in the nucleus. Proteolytic degradation of RPT6 was dependent on the localization of XopJ to the plasma membrane as well as on its catalytic triad. Mutation of the Walker B motif of RPT6 prevented XopJ-mediated degradation of the protein but not XopJ interaction. This indicates that the interaction of RPT6 with XopJ is dependent on the ATP-binding activity of RPT6, but proteolytic cleavage additionally requires its ATPase activity. Inhibition of the proteasome impairs the proteasomal turnover of Nonexpressor of Pathogenesis-Related1 (NPR1), the master regulator of salicylic acid responses, leading to the accumulation of ubiquitinated NPR1, which likely interferes with the full induction of NPR1 target genes. Our results show that YopJ family T3Es are not only highly diversified in virulence function but also appear to possess different biochemical activities. PMID:25739698

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

    PubMed

    Karmous, Inès; Jaouani, Khadija; El Ferjani, Ezzedine; Chaoui, Abdelilah

    2014-07-01

    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.

  8. Profibrillin-1 Maturation by Human Dermal Fibroblasts: Proteolytic Processing and Molecular Chaperones

    PubMed Central

    Wallis, Debra D.; Putnam, Elizabeth A.; Cretoiu, Jill S.; Carmical, Sonya G.; Cao, Shi-Nian; Thomas, Gary; Milewicz, Dianna M.

    2006-01-01

    Fibrillin-1 is synthesized as a proprotein that undergoes proteolytic processing in the unique C-terminal domain by a member of the PACE/furin family of endoproteases. This family of endoproteases is active in the trans-Golgi network (TGN), but metabolic labeling studies have been controversial as to whether profibrillin-1 is processed intracellularly or after secretion. This report provides evidence that profibrillin-1 processing is not an intracellular event. Bafilomycin A1 and incubation of dermal fibroblasts at 22°C were used to block secretion in the TGN to confirm that profibrillin-1 processing did not occur in this compartment. Profibrillin-1 immunoprecipitation studies revealed that two endoplasmic reticulum-resident molecular chaperones, BiP and GRP94, interacted with profibrillin-1. To determine the proprotein convertase responsible for processing profibrillin-1, a specific inhibitor of furin, α-1-antitrypsin, Portland variant, was both expressed in the cells and added to cells exogenously. In both cases, the inhibitor blocked the processing of profibrillin-1, providing evidence that furin is the enzyme responsible for profibrillin-1 processing. These studies delineate the secretion and proteolytic processing of profibrillin-1, and identify the proteins that interact with profibrillin-1 in the secretory pathway. PMID:14523997

  9. Hydrocarbon double-stapling remedies the proteolytic instability of a lengthy peptide therapeutic.

    PubMed

    Bird, Gregory H; Madani, Navid; Perry, Alisa F; Princiotto, Amy M; Supko, Jeffrey G; He, Xiaoying; Gavathiotis, Evripidis; Sodroski, Joseph G; Walensky, Loren D

    2010-08-10

    The pharmacologic utility of lengthy peptides can be hindered by loss of bioactive structure and rapid proteolysis, which limits bioavailability. For example, enfuvirtide (Fuzeon, T20, DP178), a 36-amino acid peptide that inhibits human immunodeficiency virus type 1 (HIV-1) infection by effectively targeting the viral fusion apparatus, has been relegated to a salvage treatment option mostly due to poor in vivo stability and lack of oral bioavailability. To overcome the proteolytic shortcomings of long peptides as therapeutics, we examined the biophysical, biological, and pharmacologic impact of inserting all-hydrocarbon staples into an HIV-1 fusion inhibitor. We find that peptide double-stapling confers striking protease resistance that translates into markedly improved pharmacokinetic properties, including oral absorption. We determined that the hydrocarbon staples create a proteolytic shield by combining reinforcement of overall alpha-helical structure, which slows the kinetics of proteolysis, with complete blockade of peptide cleavage at constrained sites in the immediate vicinity of the staple. Importantly, double-stapling also optimizes the antiviral activity of HIV-1 fusion peptides and the antiproteolytic feature extends to other therapeutic peptide templates, such as the diabetes drug exenatide (Byetta). Thus, hydrocarbon double-stapling may unlock the therapeutic potential of natural bioactive polypeptides by transforming them into structurally f