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Sample records for caspase-3-dependent proteolytic activation

  1. A potent reporter applicable to the monitoring of caspase-3-dependent proteolytic cleavage.

    PubMed

    Park, Kyoungsook; Kang, Hyo-Jin; Ahn, Junhyoung; Yi, So Yeon; Han, Sang Hee; Park, Hye-Jung; Chung, Sang J; Chung, Bong Hyun; Kim, Moonil

    2008-11-01

    In this study, we developed a chimeric caspase-3 substrate (GST:DEVD:EGFP) comprised of glutathione-S transferase (GST) and enhanced green fluorescent protein (EGFP) with a specialized linker peptide harboring the caspase-3 cleavage sequence, DEVD. Using this reporter, we assessed the proteolytic cleavage of the artificial caspase-3 substrate for caspase-3. The common feature of this approach is that the presence of the DEVD sequence between GST and EGFP allows for caspase-3-dependent cleavage after the Asp (D) residue, resulting in the elimination of EGFP from the GST:DEVD:EGFP reporter. To the best of our knowledge, this study reports the first application employing a chimeric protein substrate, with the similar accuracy level compared to the conventional methods such as fluorometric assays. As a result, using this GST:DEVD:EGFP reporter, caspase-3 activation based on proteolytic properties could be monitored via a variety of bioanalytical techniques such as immunoblot analysis, glutathione-agarose bead assay, and on-chip visualization, providing both technical and economical advantages over the extensively utilized fluorogenic peptide assay. Our results convincingly showed that this versatile reporter (GST:DEVD:EGFP) constitutes a useful system for the monitoring of caspase-3 activation, potentially enabling the monitoring of the proteolytic activities of different intra-cellular proteases via the substitution of the cleavage sequence within the same schematic construct. PMID:18775457

  2. 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.

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

    PubMed

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

    2016-09-01

    Mouse models of neurodegenerative diseases such as Alzheimer's disease (AD) are important for understanding how pathological signaling cascades change neural circuitry and with 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 with increased levels of truncated tau and formation of neurofibrillary tangles. In addition, cognitive decline was significantly correlated with increased levels of caspase activity and tau truncated by caspase-3. Experimentally induced inhibition of caspases prevented this proteolytic cleavage of tau 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

  4. 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.

  5. 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

    2016-01-01

    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

  6. 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

  7. Activation of the Nrf2 pathway, but decreased {gamma}-glutamylcysteine synthetase heavy subunit chain levels and caspase-3-dependent apoptosis during exposure of primary mouse hepatocytes to diphenylarsinic acid

    SciTech Connect

    Sumi, Daigo; Manji, Aiko; Shinkai, Yasuhiro; Toyama, Takashi; Kumagai, Yoshito

    2007-09-15

    Diphenylarsinic acid (DPAsV) is a degradation product of chemical warfare agents, over which there has been a public outcry in the Kamisu Area of Ibaraki Prefecture in Japan. In this study, we investigated the cytotoxicity of and cellular response to DPAsV in primary mouse hepatocytes. Exposure of the hepatocytes to DPAsV resulted in cell damage accompanied by cellular accumulation of DPAsV in a time-dependent manner. The cell death caused by DPAsV was attributable to apoptosis. DPAsV activated a basic leucine-zipper transcription factor Nrf2 as determined by the nuclear translocation of Nrf2, anti-oxidant response element (ARE)-dependent luciferase activity, and upregulation of downstream gene products. However, {gamma}-glutamylcysteine synthetase heavy subunit chain ({gamma}-GCS{sub H}), which is regulated by Nrf2, underwent cleavage by activated caspase-3 to a 17 kDa fragment, leading to a minimal level of constitutive {gamma}-GCS{sub H} expression 72 h following the exposure (25 {mu}M). Experiments with cycloheximide revealed that the DPAsV-mediated reduction in {gamma}-GCS{sub H} was due to a post-translational modification. The results suggest that DPAsV causes caspase-3-dependent cleavage of {gamma}-GCS{sub H} regardless of Nrf2 activation in primary mouse hepatocytes.

  8. 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

  9. 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

  10. Cucurbitacin IIa induces caspase-3-dependent apoptosis and enhances autophagy in lipopolysaccharide-stimulated RAW 264.7 macrophages.

    PubMed

    He, Jian; Wang, Yao; Xu, Li-hui; Qiao, Jing; Ouyang, Dong-yun; He, Xian-hui

    2013-05-01

    Cucurbitacin IIa (CuIIa), a member of cucurbitacin family, is isolated from the root of Hemsleya amabilis which has been used as an ancient remedy for bacillary dysentery and gastroenteritis. The anti-inflammatory properties of CuIIa have long been recognized but the underlying mechanism is largely unknown. In this study, we investigated the anti-inflammatory effect of CuIIa on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. The results showed that CuIIa inhibited the proliferation and migration of RAW 264.7 cells in a dose-dependent manner. Whereas CuIIa did not cause apoptosis in unstimulated RAW 264.7 cells, it did induce a significant apoptosis in LPS-stimulated cells, which was caspase-3-dependent and associated with downregulation of survivin. Furthermore, LPS induced autophagy in RAW 264.7 cells and this effect was further enhanced by CuIIa as evidenced by increased levels of LC3-II conjugates and formation of LC3 puncta. In addition, CuIIa disrupted actin cytoskeleton via inducing actin aggregation. However, neither the synthesis of tumor necrosis factor-α, nor the activation of the mitogen-activated protein kinases and NF-κB pathways in LPS-stimulated cells was suppressed by CuIIa treatment. Collectively, these results suggested that induction of apoptosis and enhancement of autophagy contributed to the anti-inflammatory activity of CuIIa against inflammation-related diseases. PMID:23541744

  11. Regulation of Trichophyton rubrum proteolytic activity.

    PubMed Central

    Apodaca, G; McKerrow, J H

    1989-01-01

    Trichophyton rubrum is the most common dermatophyte of humans and normally colonizes the superficial layers of the epidermis (stratum corneum). Several proteinases with a possible role in the metabolism of host proteins have been purified from this fungus. The regulation of these enzymes and their role in fungal metabolism were studied at the biochemical level. General proteolytic (azocollytic) activity was repressed when log-phase cultures of T. rubrum were grown in a minimal medium that contained readily metabolized sources of carbon, nitrogen, sulfur, and phosphorus. When either carbon, nitrogen, or sulfur was deleted from this minimal medium, azocollytic activity was derepressed. In all cases a high-molecular-weight activity (Mr, greater than 200,000) was expressed. A 71,000-Mr proteinase was observed in nitrogen-depleted cultures, and proteolytic species of Mr 124,000 and 27,000 were secreted in sulfur-depleted cultures. The addition of either inorganic (MgSO4, Na2SO3, NaS2O3) or organic (methionine, cysteine) sulfur to the sulfur-depleted medium repressed the expression of azocollytic activity. In contrast, keratinolytic activity was not repressed by carbon, nitrogen, or sulfur but instead was induced when a protein source was included in the minimal medium. Stationary-phase cultures of T. rubrum secreted all proteolytic activities constitutively. Unlike log-phase cultures, the stationary-phase cultures secreted azocollytic, elastinolytic, and keratinolytic activity in minimal medium. These activities fell in the carbon-, nitrogen-, and phosphorous-depleted media but remained high in sulfur-depleted medium. The following model is proposed for the regulation of T. rubrum proteolytic activity. In the initial stages of infection, T. rubrum grows logarithmically. In this state, proteolytic activity is derepressed whenever carbon, nitrogen, or sulfur is lacking in the fungal milieu. The general proteinases produced would act on the nonkeratinous proteins in the

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

    PubMed

    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

  13. 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

  14. 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

    2015-10-01

    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

  15. NADPH oxidase 3-associated oxidative stress and caspase 3-dependent apoptosis in the cochleae of D-galactose-induced aged rats

    PubMed Central

    DU, ZHENGDE; LI, SHUO; LIU, LIN; YANG, QIONG; ZHANG, HONGWEI; GAO, CHUNSHENG

    2015-01-01

    Oxidative damage to mitochondrial DNA (mtDNA) and cell apoptosis are heavily implicated in aging. Our previous study established a mimetic rat model of aging in the cochleae using D-galactose (D-gal), and revealed that chronic injection of D-gal can increase oxidative stress and mtDNA common deletions (CD). The aim of the present study was to investigate the sources of reactive oxygen species and the occurrence of apoptosis in the cochleae of rats following 8 weeks of D-gal exposure. The results of the present study indicated that an elevated accumulation of the mtDNA CD and mitochondrial ultrastructural damage occurred in the cochleae of rats injected with D-gal for 8 weeks. In addition, the levels of 8-hydroxy-2-deoxyguanosine, NADPH oxidase (NOX) 3, P22phox and cleaved caspase 3, and the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end-labelling-positive cells were increased in the cochleae of D-gal-treated rats, compared with the controls. These findings suggested that nitric oxide synthase NOX3-associated oxidative stress may contribute to the accumulation of mtDNA mutations and activate a caspase 3-dependent apoptotic signalling pathway in the cochleae during aging. The present study also provided novel insights into the development of age-associated hearing loss, also termed presbycusis. PMID:26498835

  16. 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

  17. 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. PMID:15978746

  18. Berberine in combination with cisplatin suppresses breast cancer cell growth through induction of DNA breaks and caspase-3-dependent apoptosis.

    PubMed

    Zhao, Yuwan; Jing, Zuolei; Li, Yan; Mao, Weifeng

    2016-07-01

    Berberine (BBR) is an isoquinoline alkaloid extracted from medicinal plants such as Hydrastis canadensis, Berberis aristata and Coptis chinensis. BBR displays a number of beneficial roles in the treatment of various types of cancers, yet the precise mechanisms of its action remain unclear. Cisplatin is an effective cancer chemotherapeutic agent and functions by generating DNA damage, promoting DNA damage-induced cell cycle arrest and apoptosis; however, its efficacy is challenged by the resistance of tumor cells in clinical application. The aim of the present study was to investigate the effects of BBR in combination with cisplatin on human breast cancer cells. MTT assay showed that BBR inhibited breast cancer MCF-7 cell growth with a 50% inhibitory concentration (IC50) value of 52.178±1.593 µM and the IC50 value of cisplatin was 49.541±1.618 µM, while in combination with 26 µM BBR, the IC50 value of cisplatin was 5.759±0.76 µM. BBR sensitized the MCF-7 cells to cisplatin in a time- and dose-dependent manner. After treatment of BBR and cisplatin, the cellular pro-apoptotic capase-3 and cleaved capspase-3 and caspase-9 were upregulated and the anti-apoptotic Bcl-2 was downregulated. Importantly, BBR restrained the expression of cellular PCNA, and immunofluoresence analysis of γH2AX showed that BBR increased the DNA damages induced by cisplatin. Taken together, the results demonstrated that BBR sensitized MCF-7 cells to cisplatin through induction of DNA breaks and caspase-3-dependent apoptosis. PMID:27177238

  19. Activation and Proteolytic Activity of the Treponema pallidum Metalloprotease, Pallilysin

    PubMed Central

    Houston, Simon; Hof, Rebecca; Honeyman, Lisa; Hassler, Julia; Cameron, Caroline E.

    2012-01-01

    Treponema pallidum is a highly invasive pathogen that undergoes rapid dissemination to establish widespread infection. Previous investigations identified the T. pallidum adhesin, pallilysin, as an HEXXH-containing metalloprotease that undergoes autocatalytic cleavage and degrades laminin and fibrinogen. In the current study we characterized pallilysin's active site, activation requirements, cellular location, and fibrin clot degradation capacity through both in vitro assays and heterologous treponemal expression and degradation studies. Site-directed mutagenesis showed the pallilysin HEXXH motif comprises at least part of the active site, as introduction of three independent mutations (AEXXH [H198A], HAXXH [E199A], and HEXXA [H202A]) abolished pallilysin-mediated fibrinogenolysis but did not adversely affect host component binding. Attainment of full pallilysin proteolytic activity was dependent upon autocatalytic cleavage of an N-terminal pro-domain, a process which could not occur in the HEXXH mutants. Pallilysin was shown to possess a thrombin cleavage site within its N-terminal pro-domain, and in vitro studies confirmed cleavage of pallilysin with thrombin generates a truncated pallilysin fragment that has enhanced proteolytic activity, suggesting pallilysin can also exploit the host coagulation process to facilitate protease activation. Opsonophagocytosis assays performed with viable T. pallidum demonstrated pallilysin is a target of opsonic antibodies, consistent with a host component-interacting, surface-exposed cellular location. Wild-type pallilysin, but not the HEXXA mutant, degraded fibrin clots, and similarly heterologous expression of pallilysin in the non-invasive spirochete Treponema phagedenis facilitated fibrin clot degradation. Collectively these results identify pallilysin as a surface-exposed metalloprotease within T. pallidum that possesses an HEXXH active site motif and requires autocatalytic or host-mediated cleavage of a pro-domain to attain

  20. 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

  1. Silk microgels formed by proteolytic enzyme activity.

    PubMed

    Samal, Sangram K; Dash, Mamoni; Chiellini, Federica; Kaplan, David L; Chiellini, Emo

    2013-09-01

    The proteolytic enzyme α-chymotrypsin selectively cleaves the amorphous regions of silk fibroin protein (SFP) and allows the crystalline regions to self-assemble into silk microgels (SMGs) at physiological temperature. These microgels consist of lamellar crystals in the micrometer scale, in contrast to the nanometer-scaled crystals in native silkworm fibers. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zeta potential results demonstrated that α-chymotrypsin utilized only the non-amorphous domains or segments of the heavy chain of SFP to form negatively charged SMGs. The SMGs were characterized in terms of size, charge, structure, morphology, crystallinity, swelling kinetics, water content and thermal properties. The results suggest that the present technique of preparing SMGs by α-chymotrypsin is simple and efficient, and that the prepared SMGs have useful features for studies related to biomaterial and pharmaceutical needs. This process is also an easy way to obtain the amorphous peptide chains for further study. PMID:23756227

  2. Multidrug resistance-associated protein 3 confers resistance to chemoradiotherapy for rectal cancer by regulating reactive oxygen species and caspase-3-dependent apoptotic pathway.

    PubMed

    Yu, Zhiqi; Zhang, Chang; Wang, Hao; Xing, Junjie; Gong, Haifeng; Yu, Enda; Zhang, Wei; Zhang, Xiaoqing; Cao, Guangwen; Fu, Chuangang

    2014-10-28

    This study aimed to clarify the role of multidrug resistance-associated protein 3 (MRP3) in resistance to neoadjuvant chemoradiotherapy and long-term prognosis of advanced rectal cancer. Immunohistochemistry was used to measure MRP3 expression in biopsy specimens of 144 stage II-III rectal cancer patients who received preoperative chemoradiotherapy. The effect of MRP3 expression on short-term pathological response and postoperative long-term prognosis were assessed using the Cox proportional hazards model. Short interfering RNAs targeting MRP3 were synthesized and used to transfect human colorectal carcinoma cell lines. The effect of MRP3 down-regulation on cell proliferation and apoptosis in response to 5-fluorouracil and/or irradiation were examined in vitro and in xenograft mouse models, respectively. The content of intracellular reactive oxygen species and the activity of caspase-3-dependent apoptotic pathway in response to irradiation were further evaluated. High expression (immunoreactive score > 6) of MRP3 significantly predicted poor pathological response to chemoradiotherapy (tumor regression grade ≤ 2 vs. ≥3, p = 0.002) in univariate analysis and unfavorable long-term prognosis (5-year overall survival: HR = 1.612, 95% CI, 1.094-2.375, p = 0.016; 5-year disease-free survival: HR = 1.513, 95% CI, 1.041-2.200, p = 0.030) in multivariate Cox analysis. MRP3 down-regulation significantly increased 5-fluorouracil or irradiation-induced cell apoptosis and attenuated tumor growth following irradiation in animal models. MRP3 inhibition significantly reduced intracellular reactive oxygen species exporting from cells following irradiation, and increased expression of cleaved poly ADP-ribose polymerase and caspase-3. Aberrant expression of MRP3 in rectal cancer confers chemo-radioresistance. MRP3 might be a predictive factor and an attractive target in treating advanced rectal cancer. PMID:25088576

  3. Proteolytic activation defines distinct lymphangiogenic mechanisms for VEGFC and VEGFD

    PubMed Central

    Bui, Hung M.; Enis, David; Robciuc, Marius R.; Nurmi, Harri J.; Cohen, Jennifer; Chen, Mei; Yang, Yiqing; Dhillon, Veerpal; Johnson, Kathy; Zhang, Hong; Kirkpatrick, Robert; Traxler, Elizabeth; Alitalo, Kari

    2016-01-01

    Lymphangiogenesis is supported by 2 homologous VEGFR3 ligands, VEGFC and VEGFD. VEGFC is required for lymphatic development, while VEGFD is not. VEGFC and VEGFD are proteolytically cleaved after cell secretion in vitro, and recent studies have implicated the protease a disintegrin and metalloproteinase with thrombospondin motifs 3 (ADAMTS3) and the secreted factor collagen and calcium binding EGF domains 1 (CCBE1) in this process. It is not well understood how ligand proteolysis is controlled at the molecular level or how this process regulates lymphangiogenesis, because these complex molecular interactions have been difficult to follow ex vivo and test in vivo. Here, we have developed and used biochemical and cellular tools to demonstrate that an ADAMTS3-CCBE1 complex can form independently of VEGFR3 and is required to convert VEGFC, but not VEGFD, into an active ligand. Consistent with these ex vivo findings, mouse genetic studies revealed that ADAMTS3 is required for lymphatic development in a manner that is identical to the requirement of VEGFC and CCBE1 for lymphatic development. Moreover, CCBE1 was required for in vivo lymphangiogenesis stimulated by VEGFC but not VEGFD. Together, these studies reveal that lymphangiogenesis is regulated by two distinct proteolytic mechanisms of ligand activation: one in which VEGFC activation by ADAMTS3 and CCBE1 spatially and temporally patterns developing lymphatics, and one in which VEGFD activation by a distinct proteolytic mechanism may be stimulated during inflammatory lymphatic growth. PMID:27159393

  4. 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. PMID:23454496

  5. 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.

  6. 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. PMID:26511318

  7. 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. PMID:22412883

  8. 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

  9. 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

  10. 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

  11. Single cell multiplexed assay for proteolytic activity using droplet microfluidics.

    PubMed

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

    2016-07-15

    Cellular enzymes interact in a post-translationally regulated fashion to govern individual cell behaviors, yet current platform technologies are limited in their ability to measure multiple enzyme activities simultaneously in single cells. Here, we developed multi-color Förster resonance energy transfer (FRET)-based enzymatic substrates and use them in a microfluidics platform to simultaneously measure multiple specific protease activities from water-in-oil droplets that contain single cells. By integrating the microfluidic platform with a computational analytical method, Proteolytic Activity Matrix Analysis (PrAMA), we are able to infer six different protease activity signals from individual cells in a high throughput manner (~100 cells/experimental run). We characterized protease activity profiles at single cell resolution for several cancer cell lines including breast cancer cell line MDA-MB-231, lung cancer cell line PC-9, and leukemia cell line K-562 using both live-cell and in-situ cell lysis assay formats, with special focus on metalloproteinases important in metastasis. The ability to measure multiple proteases secreted from or expressed in individual cells allows us to characterize cell heterogeneity and has potential applications including systems biology, pharmacology, cancer diagnosis and stem cell biology. PMID:26995287

  12. Determination of lipolytic and proteolytic activities of mycoflora isolated from dry-cured teruel ham.

    PubMed

    Alapont, C; Martínez-Culebras, P V; López-Mendoza, M C

    2015-08-01

    Fungi play a key role in dry-cured ham production because of their lipolytic and proteolytic activities. In the present study, 74 fungal strains from dry-cured Teruel hams and air chambers were tested for proteolytic and lipolytic activities, with a view to their possible use as starter cultures. Lipolytic activity of fungi was studied against lauric, palmitic, stearic and oleic acids, whereas proteolytic activity was studied against casein and myosin. Of the 74 fungal strains tested, most of them demonstrated lipolytic activity (94.59 %). Lipolytic activity against lauric and oleic acids was stronger than against palmitic and stearic acids. 39 strains (52.70 %) demonstrated proteolytic activity against casein and the 6 highest proteolytic strains were also tested for pork myosin proteolysis. Some strains belonging to Penicillium commune, Penicillium chrysogenum, Penicillium nalgiovense and Cladosporium cladosporioides were selected because of their significant proteolytic and lipolytic activities and could be suitable to use as starters in dry-cured ham. PMID:26243949

  13. When activity requires breaking up: LEKTI proteolytic activation cascade for specific proteinase inhibition.

    PubMed

    Furio, Laetitia; Hovnanian, Alain

    2011-11-01

    Lymphoepithelial Kazal-type related inhibitor (LEKTI) is a multidomain proteinase inhibitor whose defective expression causes Netherton syndrome (NS). LEKTI is encoded by SPINK5, which is also a susceptibility gene for atopic disease. In this issue, Fortugno et al. report an elegant and thorough study of the LEKTI proteolytic activation process in which they identify the precise nature of the cleavage sites used and the bioactive fragments generated. They propose a proteolytic activation model in human skin and confirm differential inhibition of kallikrein (KLK) 5, 7, and 14 by the major physiological LEKTI fragments. They show that these bioactive fragments inhibit KLK-mediated proteolysis of desmoglein 1 (DSG1) and suggest a fine-tuned inhibition process controlling target serine proteinase (SP) activity. PMID:21997416

  14. Comparison of the growth kinetics and proteolytic activities of Chryseobacterium species and Pseudomonas fluorescens.

    PubMed

    Bekker, A; Steyn, L; Charimba, G; Jooste, P; Hugo, C

    2015-12-01

    The effect of temperature on the growth kinetics and proteolytic activity of Chryseobacterium joostei and Chryseobacterium bovis was determined during this study. The results were compared with the activities of Pseudomonas fluorescens, which is regarded to be a major food spoilage psychrotolerant microorganism. For the growth studies, cultures were incubated in nutrient broth in a temperature gradient incubator (from 9 to 50 °C) and separately at 4 °C, and the optical density was measured at different time intervals. Growth temperature profiles for each organism were constructed. For determination of proteolytic activity, the cultures were incubated in fat-free ultra-high temperature processed milk in the temperature gradient incubator for 72 h (temperature range as above). Cell-free extracts were used to determine the proteolytic activity using the azocasein method. Results of the growth studies showed that C. joostei had the ability to grow over a wider temperature range than C. bovis and P. fluorescens without being affected by changes in the temperature. For the proteolytic activity, C. joostei had significantly (p < 0.001) higher activity per milligram of protein at 15.5 °C, followed by C. bovis and P. fluorescens. The results showed that C. joostei potentially has an even greater spoilage capacity in milk on the basis of growth rate and proteolytic activity than did P. fluorescens. PMID:26451905

  15. 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

  16. 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.

  17. Proteolytic activity regarding Sarconesiopsis magellanica (Diptera: Calliphoridae) larval excretions and secretions.

    PubMed

    Pinilla, Yudi T; Moreno-Pérez, Darwin A; Patarroyo, Manuel A; Bello, Felio J

    2013-12-01

    Sarconesiopsis magellanica (Diptera: Calliphoridae) is a medically important necrophagous fly which is used for establishing the post-mortem interval. Diptera maggots release proteolytic enzymes contained in larval excretion and secretion (ES) products playing a key role in digestion. Special interest in proteolytic enzymes has also been aroused regarding understanding their role in wound healing since they degrade necrotic tissue during larval therapy. This study was thus aimed at identifying and characterising S. magellanica proteolytic enzyme ES products for the first time. These products were obtained from first-, second- and third-instar larvae taken from a previously-established colony. ES proteins were separated by SDS-PAGE and their proteolytic activity was characterised by zymograms and inhibition assays involving BAPNA (Nα-benzoyl-dl-Arg-p-nitroanilide) and SAPNA substrates, using synthetic inhibitors. The protein profile ranged from ∼69kDa to ∼23kDa; several of them coincided with the Lucilia sericata ES protein profile. Serine-protease hydrolysis activity (measured by zymogram) was confirmed when a ∼25kDa band disappeared upon ES incubation with PMSF inhibitor at pH 7.8. Analysis of larval ES proteolytic activity on BAPNA and SAPNA substrates (determined by using TLCK and TPCK specific inhibitors) suggested a greater amount of trypsin-like protease. These results support the need for further experiments aimed at validating S. magellanica use in larval therapy. PMID:24076089

  18. 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

  19. Proteolytic activity of molds and their metabiotic association with Salmonella in a model system.

    PubMed

    Cibelli, F; Ciccarone, C; Altieri, C; Bevilacqua, A; Sinigaglia, M

    2008-10-01

    The aim of this study was to investigate the proteolytic ability of some strains of aspergilli, fusaria, and penicillia and the metabiotic effect of Fusarium oxysporum and Penicillium expansum on Salmonella. The proteolytic activity of the target molds was determined on tomato juice agar and tomato juice, whereas the metabiotic effect of F. oxysporum and P. expansum on Salmonella was assessed in a model system consisting of tryptone soy broth with different amounts of tomato juice added. Fusaria, some aspergilli, and one strain of penicillium showed a proteolytic activity on tomato juice agar. In addition, Salmonella survival was enhanced in tryptone soy broth plus 20 or 50% tomato juice in the model system previously inoculated with F. oxysporum. PMID:18939766

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

    PubMed Central

    Atanasova, Jivka; Moncheva, Penka; Ivanova, Iskra

    2014-01-01

    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. PMID:26019593

  1. Proteolytic activation of latent Paraguaya peach PPO. Characterization of monophenolase activity.

    PubMed

    Laveda, F; Núñez-Delicado, E; García-Carmona, F; Sánchez-Ferrer, A

    2001-02-01

    The kinetics of the activation process of latent peach PPO by trypsin was studied. By coupling this activation process to the oxidation of 4-tert-butylcatechol (TBC) to its corresponding quinone, it was possible to evaluate the specific rate constant of active PPO formation, k(3), which showed a value of 0.04 s(-1). This proteolytic activation of latent peach PPO permitted us to characterize the monophenolase activity of peach PPO for the first time using p-cresol as substrate, and it showed the characteristic lag period of the kinetic mechanism of monophenols hydroxylation, which depended on the enzyme and substrate concentration, the pH and the presence of catalytic amounts of o-diphenol (4-methylcatechol). The enzyme activation constant, k(act), was 2 microM. PMID:11262063

  2. 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.

  3. Thrombin stimulates fibroblast procollagen production via proteolytic activation of protease-activated receptor 1.

    PubMed Central

    Chambers, R C; Dabbagh, K; McAnulty, R J; Gray, A J; Blanc-Brude, O P; Laurent, G J

    1998-01-01

    Thrombin is a multifunctional serine protease that has a crucial role in blood coagulation. It is also a potent mesenchymal cell mitogen and chemoattractant and might therefore have an important role in the recruitment and local proliferation of mesenchymal cells at sites of tissue injury. We hypothesized that thrombin might also affect the deposition of connective tissue proteins at these sites by directly stimulating fibroblast procollagen production. To address this hypothesis, the effect of thrombin on procollagen production and gene expression by human foetal lung fibroblasts was assessed over 48 h. Thrombin stimulated procollagen production at concentrations of 1 nM and above, with maximal increases of between 60% and 117% at 10 nM thrombin. These effects of thrombin were, at least in part, due to increased steady-state levels of alpha1(I) procollagen mRNA. They could furthermore be reproduced with thrombin receptor-activating peptides for the protease-activated receptor 1 (PAR-1) and were completely abolished when thrombin was rendered proteolytically inactive with the specific inhibitors d-Phe-Pro-ArgCH2Cl and hirudin, indicating that thrombin is mediating these effects via the proteolytic activation of PAR-1. These results suggest that thrombin might influence the deposition of connective tissue proteins during normal wound healing and the development of tissue fibrosis by stimulating fibroblast procollagen production. PMID:9639571

  4. 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. PMID:21198710

  5. Extracellular thermostable proteolytic activity of the milk spoilage bacterium Pseudomonas fluorescens PS19 on bovine caseins.

    PubMed

    Stuknytė, M; Decimo, M; Colzani, M; Silvetti, T; Brasca, M; Cattaneo, S; Aldini, G; De Noni, I

    2016-06-01

    We studied the thermostable proteolytic activity of Pseudomonas fluorescens PS19 isolated from raw bovine milk. The heat-treated cell-free supernatant (HT-CFS) contained a thermostable protease of approximately 45 kDa, as revealed by casein zymography. We assigned this enzyme to P. fluorescens AprX metalloprotease (UniProtKB Acc. No. C9WKP6). After concentration by ultrafiltration at 10 kDa, the HT-CFS showed 2 other thermostable proteolytic bands on zymogram, with molecular masses of approximately 15 and 25 kDa. The former resulted a fragment of the AprX protease, whereas the 25-kDa protease was not homologous to any known protein of Pseudomonas spp. Subsequently, we assessed the proteolytic activity of the HT-CFS on bovine αS-, β-, and κ-casein during in vitro incubation at 7 or 22°C. By means of ultra-performance liquid chromatography-tandem mass spectrometry we identified the released peptides (n=591). Some of them resisted proteolysis during the whole incubation period at both incubation temperatures and, therefore, they could be assumed as indicators of the proteolytic action of P. fluorescens PS19 on bovine caseins. PMID:26995139

  6. 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. PMID:26529648

  7. [Activity of proteolytic and nucleolytic enzymes from the gonades of hydrobionts].

    PubMed

    Pozdniakova, Iu M; Pivnenko, T N; Epshteĭn, L M

    2004-01-01

    The activity of nucleolytic and proteolytic enzymes in milt of nine kinds of fishes belonging to various families and of three kinds invertebrates is determined. There is carried out electrophoreses division of preparations DNA, received from milts by various methods; there are determined structure and molecular weights of oligonucleotides. The influence of activity tissue enzymes on a destruction degree of DNA is established at addition enzymes of exogenic origin. PMID:19621738

  8. 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.

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

    PubMed Central

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

    1993-01-01

    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 AI) 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(r)) 15,000 to 18,000. We 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, we found that antibodies to alpha AI recognize large (M(r) 30,000-35,000) polypeptides as well as typical alpha AI processing products (M(r) 15,000-18,000). Alpha AI activity was found in all extracts that had the typical alpha AI processed polypeptides, but was absent from seed extracts that lacked such polypeptides. Second, we made a mutant alpha AI in which asparagine-77 is changed to aspartic acid-77. This mutation slows down the proteolytic processing of pro-alpha AI when the gene is expressed in tobacco. When pro-alpha AI was separated from mature alpha AI by gel filtration, pro-alpha AI was found not to have alpha-amylase inhibitory activity. We interpret these results to mean that formation of the active inhibitor is causally related to proteolytic processing of the proprotein. We suggest that the polypeptide cleavage removes a conformational constraint on the precursor to produce the biochemically active molecule. PMID:8310064

  10. [A micromethod for the rapid detection of proteolytic activity of microorganisms].

    PubMed

    Kuznetsova, G G

    1989-01-01

    The author has developed a micromodification of the rapid photoemulsion method for the detection of the microorganism proteolytic activity. The test is carried out on polystyrene plates, meat-peptone broth is used as the suspension fluid. Rectangular film strips are vertically placed into the wells, thus preventing false-negative reactions possible in case of an erroneous horizontal position of the film with the emulsion layer turned upwards if square or round film fragments are used. The proteolytic activities of 120 microorganism strains (95 of these hydrolyze gelatin) have been examined by the micromethod and the routine test with gelatin. The suggested test is 2-4-fold more rapid than the routine one used in investigations of the cultures slowly hydrolyzing gelatin. The fact that the test is carried out on the plates considerably reduces the nutrient medium and the number of laboratory glassware and helps obtain more accurate results. PMID:2468029

  11. In vivo sensing of proteolytic activity with an NSET-based NIR fluorogenic nanosensor.

    PubMed

    Ku, Minhee; Hong, Yoochan; Heo, Dan; Lee, Eugene; Hwang, Seungyeon; Suh, Jin-Suck; Yang, Jaemoon

    2016-03-15

    Biomedical in vivo sensing methods in the near-infrared (NIR) range, which that provide relatively high photon transparency, separation from auto-fluorescence background, and extended sensitivity, are being used increasingly for non-invasive mapping and monitoring of molecular events in cancer cells. In this study, we fabricated an NIR fluorogenic nanosensor based on the nanoparticle surface energy transfer effect, by conjugation of fluorescent proteolytic enzyme-specific cleavable peptides with gold nanorods (GNRs). Membrane-anchored membrane type 1-matrix metalloproteinases (MT1-MMPs), a family of zinc-dependent proteolytic enzymes, can induce the metastatic potential of cancer cells by promoting degradation of the extracellular matrix. Therefore, sensitive detection of MT1-MMP activity can provide essential information in the clinical setting. We have applied in vivo NIR sensing to evaluate MT1-MMP activity, as an NIR imaging target, in an MT1-MMP-expressing metastatic tumor mouse model. PMID:26454829

  12. 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. PMID:26852027

  13. 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

  14. 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

  15. Circulating FGF21 proteolytic processing mediated by fibroblast activation protein

    PubMed Central

    Zhen, Eugene Y.; Jin, Zhaoyan; Ackermann, Bradley L.; Thomas, Melissa K.; Gutierrez, Jesus A.

    2015-01-01

    Fibroblast growth factor 21 (FGF21), a hormone implicated in the regulation of glucose homoeostasis, insulin sensitivity, lipid metabolism and body weight, is considered to be a promising therapeutic target for the treatment of metabolic disorders. Despite observations that FGF21 is rapidly proteolysed in circulation rending it potentially inactive, little is known regarding mechanisms by which FGF21 protein levels are regulated. We systematically investigated human FGF21 protein processing using mass spectrometry. In agreement with previous reports, circulating human FGF21 was found to be cleaved primarily after three proline residues at positions 2, 4 and 171. The extent of FGF21 processing was quantified in a small cohort of healthy human volunteers. Relative abundance of FGF21 proteins cleaved after Pro-2, Pro-4 and Pro-171 ranged from 16 to 30%, 10 to 25% and 10 to 34%, respectively. Dipeptidyl peptidase IV (DPP-IV) was found to be the primary protease responsible for N-terminal cleavages after residues Pro-2 and Pro-4. Importantly, fibroblast activation protein (FAP) was implicated as the protease responsible for C-terminal cleavage after Pro-171, rendering the protein inactive. The requirement of FAP for FGF21 proteolysis at the C-terminus was independently demonstrated by in vitro digestion, immunodepletion of FAP in human plasma, administration of an FAP-specific inhibitor and by human FGF21 protein processing patterns in FAP knockout mouse plasma. The discovery that FAP is responsible for FGF21 inactivation extends the FGF21 signalling pathway and may enable novel approaches to augment FGF21 actions for therapeutic applications. PMID:26635356

  16. 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

  17. 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

  18. Dual inhibition of MEK1/2 and EGFR synergistically induces caspase-3-dependent apoptosis in EGFR inhibitor-resistant lung cancer cells via BIM upregulation.

    PubMed

    Song, Ji-Young; Kim, Choung-Soo; Lee, Je-Hwan; Jang, Se Jin; Lee, Sang-wook; Hwang, Jung Jin; Lim, Chulsoo; Lee, Gilnam; Seo, Jeongbeob; Cho, Suk Young; Choi, Jene

    2013-12-01

    Epidermal growth factor receptor (EGFR) gene mutations activate the KRAS-RAF-MEK-ERK pathway in lung cancer cells. EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib induce apoptosis of cancer cells, but prolonged treatment is often associated with acquired resistance. Here, we identified a novel MEK1/2 inhibitor, CZ0775, and compared its cytotoxic effects to those of AZD6244 (selumetinib) in non-small cell lung cancer (NSCLC) cell lines harboring EGFR mutations. The lapatinib-sensitive HCC827 and PC9 and lapatinib-resistant H1650 and H1975 cell lines showed poor responses to CZ0775 and AZD6244 monotherapy with an IC50 > 10 μM. By contrast, combination treatment with lapatinib and CZ0775 inhibited cell proliferation and produced a 2-fold higher number of annexin V-labeled cells than lapatinib alone in H1975 cells. Furthermore, combination treatment decreased phosphorylated extracellular signal related kinase (p-ERK) and survivin levels and upregulated the expression of the pro-apoptotic protein BIM. siRNA-mediated BIM depletion reduced caspase-3 activity (~40%) in lapatinib and CZ0775 treated H1975 cells. An in vitro ERK activity assay showed that p-ERK levels were approximately a 3-fold lower in H1975 cells treated with CZ0775 and lapatinib combination than in cells treated with lapatinib alone. CZ0775 was more cytotoxic than AZD6244 when used in combination with lapatinib. Our results suggest that combination treatment with CZ0774 and EGFR inhibitors is a promising therapeutic approach for the treatment of EGFR-TKI-resistant lung cancers and its effect is mediated by the inhibition of ERK and the induction of BIM. PMID:24068620

  19. Chlorpyrifos Induces MLL Translocations Through Caspase 3-Dependent Genomic Instability and Topoisomerase II Inhibition in Human Fetal Liver Hematopoietic Stem Cells.

    PubMed

    Lu, Chengquan; Liu, Xiaohui; Liu, Chang; Wang, Jian; Li, Chunna; Liu, Qi; Li, Yachen; Li, Shuangyue; Sun, Shu; Yan, Jinsong; Shao, Jing

    2015-10-01

    Household pesticide exposure during pregnancy has been associated with a more than 2-fold increased risk in infant leukemia, and chlorpyrifos (CPF) is among the most frequently applied insecticides. During early fetal development, liver is a hematopoietic organ with majority of cells being CD34(+) hematopoietic stem cells (CD34(+)HSC). The in utero injury to CD34(+)HSC has been known to underlie the pathogenesis of several blood disorders, often involving rearrangements of the mixed-lineage leukemia (MLL) gene on 11q23. In this study, we evaluated the leukemogenic potential of CPF in human fetal liver-derived CD34(+)HSC. Specifically, exposure to 10 μM CPF led to decrease in viability, inhibition in proliferation and induction of DNA double-strand breaks (DSBs) and occurrence of MLL(+) rearrangements. In particular, we observed CPF-mediated cell cycle disturbance as shown by G0/G1 arrest, in contrast to etoposide (VP-16), an anticancer drug used as a positive control and known to induce G2/M arrest. Further study on mechanisms underlying DNA DSBs and MLL(+) rearrangements revealed that CPF might act as topoisomerase II poison, a mechanism of action similar to VP-16. On the other hand, CPF was also shown to induce early apoptosis through active caspase-3 activation, a pathway known to underlie DNA DSBs and MLL(+) translocations. Our data indicate that in utero injury of CD34(+)HSC by CPF may contribute to the increased risk of infant leukemia. Future work will elucidate the mechanism and the type of CPF-induced MLL(+) translocations in HSC. PMID:26198043

  20. Losac, the First Hemolin that Exhibits Procogulant Activity through Selective Factor X Proteolytic Activation*

    PubMed Central

    Alvarez-Flores, Miryam Paola; Furlin, Daniel; Ramos, Oscar H. P.; Balan, Andrea; Konno, Katsuhiro; Chudzinski-Tavassi, Ana Marisa

    2011-01-01

    Envenoming by the contact of human skin with Lonomia obliqua caterpillars promotes a hemorrhagic syndrome characterized by a consumptive coagulopathy. Losac (Lonomia obliqua Stuart factor activator) is a component of the bristle of L. obliqua that is probably partially responsible for the observed syndrome because it activates factor X and is recognized by an effective antilonomic serum. Here we unveil the proteolytic activity of Losac and demonstrate the feasibility of its recombinant production. On the other hand, Losac has no homology to known proteases, but it can be inhibited by PMSF, a serine protease inhibitor. Instead, it shows closer homology to members of the hemolin family of proteins, a group of cell adhesion molecules. The recombinant protein (rLosac) shortened the coagulation time of normal and deficient plasmas, whereas it was ineffective in factor X-deficient plasma unless reconstituted with this protein. rLosac was able to activate factor X in a dose- and time-dependent manner but not γ-carboxyglutamic acid domainless factor X. Moreover, phospholipids and calcium ions increased rLosac activity. Also, rLosac had no effect on fibrin or fibrinogen, indicating its specificity for blood coagulation activation. Linear double reciprocal plots indicate that rLosac follows a Michaelis-Menten kinetics. Cleavage of factor X by rLosac resulted in fragments that are compatible with those generated by RVV-X (a well known factor X activator). Together, our results validate Losac as the first protein from the hemolin family exhibiting procoagulant activity through selective proteolysis on coagulation factor X. PMID:21177860

  1. Monoclonal Antibodies that Inhibit the Proteolytic Activity of Botulinum Neurotoxin Serotype/B.

    PubMed

    Fan, Yongfeng; Dong, Jianbo; Lou, Jianlong; Wen, Weihua; Conrad, Fraser; Geren, Isin N; Garcia-Rodriguez, Consuelo; Smith, Theresa J; Smith, Leonard A; Ho, Mengfei; Pires-Alves, Melissa; Wilson, Brenda A; Marks, James D

    2015-09-01

    Existing antibodies (Abs) used to treat botulism cannot enter the cytosol of neurons and bind to botulinum neurotoxin (BoNT) at its site of action, and thus cannot reverse paralysis. However, Abs targeting the proteolytic domain of the toxin could inhibit the proteolytic activity of the toxin intracellularly and potentially reverse intoxication, if they could be delivered intracellularly. As such, antibodies that neutralize toxin activity could serve as potent inhibitory cargos for therapeutic antitoxins against botulism. BoNT serotype B (BoNT/B) contains a zinc endopeptidase light chain (LC) domain that cleaves synaoptobrevin-2, a SNARE protein responsible for vesicle fusion and acetylcholine vesicle release. To generate monoclonal Abs (mAbs) that could reverse paralysis, we targeted the protease domain for Ab generation. Single-chain variable fragment (scFv) libraries from immunized mice or humans were displayed on yeast, and 19 unique BoNT/B LC-specific mAbs isolated by fluorescence-activated cell sorting (FACS). The equilibrium dissociation constants (KD) of these mAbs for BoNT/B LC ranged from 0.24 nM to 14.3 nM (mean KD 3.27 nM). Eleven mAbs inhibited BoNT/B LC proteolytic activity. The fine epitopes of selected mAbs were identified by alanine-scanning mutagenesis, revealing that inhibitory mAbs bound near the active site, substrate-binding site or the extended substrate-binding site. The results provide mAbs that could prove useful for intracellular reversal of paralysis and identify epitopes that could be targeted by small molecules inhibitors. PMID:26343720

  2. Monoclonal Antibodies that Inhibit the Proteolytic Activity of Botulinum Neurotoxin Serotype/B

    PubMed Central

    Fan, Yongfeng; Dong, Jianbo; Lou, Jianlong; Wen, Weihua; Conrad, Fraser; Geren, Isin N.; Garcia-Rodriguez, Consuelo; Smith, Theresa J.; Smith, Leonard A.; Ho, Mengfei; Pires-Alves, Melissa; Wilson, Brenda A.; Marks, James D.

    2015-01-01

    Existing antibodies (Abs) used to treat botulism cannot enter the cytosol of neurons and bind to botulinum neurotoxin (BoNT) at its site of action, and thus cannot reverse paralysis. However, Abs targeting the proteolytic domain of the toxin could inhibit the proteolytic activity of the toxin intracellularly and potentially reverse intoxication, if they could be delivered intracellularly. As such, antibodies that neutralize toxin activity could serve as potent inhibitory cargos for therapeutic antitoxins against botulism. BoNT serotype B (BoNT/B) contains a zinc endopeptidase light chain (LC) domain that cleaves synaoptobrevin-2, a SNARE protein responsible for vesicle fusion and acetylcholine vesicle release. To generate monoclonal Abs (mAbs) that could reverse paralysis, we targeted the protease domain for Ab generation. Single-chain variable fragment (scFv) libraries from immunized mice or humans were displayed on yeast, and 19 unique BoNT/B LC-specific mAbs isolated by fluorescence-activated cell sorting (FACS). The equilibrium dissociation constants (KD) of these mAbs for BoNT/B LC ranged from 0.24 nM to 14.3 nM (mean KD 3.27 nM). Eleven mAbs inhibited BoNT/B LC proteolytic activity. The fine epitopes of selected mAbs were identified by alanine-scanning mutagenesis, revealing that inhibitory mAbs bound near the active site, substrate-binding site or the extended substrate-binding site. The results provide mAbs that could prove useful for intracellular reversal of paralysis and identify epitopes that could be targeted by small molecules inhibitors. PMID:26343720

  3. Investigation of plant latices of Asteraceae and Campanulaceae regarding proteolytic activity.

    PubMed

    Sytwala, Sonja; Domsalla, André; Melzig, Matthias F

    2015-12-01

    Occurrence of plant latices is widespread, there are more than 40 families of plants characterized to establish lactiferous structures. The appearance of hydrolytic active proteins, incorporated in latices is already characterized, and hydrolytic active proteins are considerable, and for several plant families, the occurrence of hydrolytic active proteins is already specified e.g. Apocynaceae Juss., Caricaceae Dumort, Euphorbiaceae Juss., Moraceae Gaudich and Papaveraceae Juss. In our investigation, focused on latex bearing plants of order Asterales, Asteraceae and Campanulaceae in particular. The present outcomes represent a comprehensive study, relating to the occurrence of proteolytic active enzymes of order Asterales for the first time. 131 different species of Asteraceae and Campanulaceae were tested, and the appearance of plant latex proteases were determined in different quantities. Proteolytic activity was investigated by inhibitory studies and determination of residual activity in the following, enable us to characterize the proteases. Most of the considered species exhibit a serine protease activity and a multiplicity of species exhibited two or more subclasses of proteases. PMID:26458257

  4. 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. PMID:26608308

  5. Proteolytic activity of Oenococcus oeni enables the increase in antioxidant and antihypertensive activities from wine.

    PubMed

    Apud, Gisselle Raquel; Stivala, María Gilda; Fernández, Pedro Aredes; Rodríguez Vaquero, María José

    2013-01-01

    Oenococcus oeni is a lactic acid bacterium involved in winemaking where it generally carries out the malolactic fermentation converting the wine's malic acid into lactic acid. In this work were used the strain m of Oenococcus oeni. The culture was inoculated at 10⁸ Log CFU/mL in a synthetic wine medium (SW) supplemented with a fraction of high molecular weight constituted by proteins and polypeptides (FPP) obtained from Cabernet Sauvignon and Syrah wines from Colalao del Valle, Tucumán, Argentine. In presence of FPP, O. oeni maintains viability after 48 h incubation time and release an extracellular proteolytic activity. Therefore, a release peptides of 1.247 and 1.373 mg N/L at 48 h of incubation time was detected in SW supplemented with FPP from Cabernet Sauvignon and Syrah wines respectively. Concomitantly with the maximum peptide release, the "in vitro" biological activities were increased. The released peptides from Cabernet Sauvignon wine enables the increase in the ferric reducing antioxidant power (FRAP) capacity, the scavenging activity of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), and the inhibition of angiotensin I-converting enzyme (ACEI activity) in 392.8 µmol FeSO₄/L, 9.7% and 63.9%, respectively. In presence of FPP of Syrah wine, the released peptides increases in 156.5 µmol FeSO₄/L, 5.5% and 13.8% the FRAP, DPPH and ACEI activities, respectively. The utilization of Oenococcus oeni m to carry out the malolactic fermentation would contribute to enhance the beneficial biological activities of the final product and provide an additional value to regional wines. PMID:24372266

  6. 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

  7. 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. PMID:26922385

  8. 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

  9. 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. PMID:26273250

  10. 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

  11. 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

  12. Effect of midgut proteolytic activity on susceptibility of lepidopteran larvae to Bacillus thuringiensis subsp. Kurstaki

    PubMed Central

    Talaei-Hassanloui, Reza; Bakhshaei, Raziyeh; Hosseininaveh, Vahid; Khorramnezhad, Ayda

    2014-01-01

    Bacillus thuringiensis (Bt) is the most effective microbial control agent for controlling numerous species from different insect orders. All subspecies and strains of B. thuringiensis can produce a spore and a crystalline parasporal body. This crystal which contains proteinaceous protoxins is dissolved in the alkaline midgut, the resulting molecule is then cleaved and activated by proteolytic enzymes and acts as a toxin. An interesting aspect of this activation process is that variations in midgut pH and protease activity have been shown to account for the spectrum of some Bt proteins activity. Thus, an important factor that could be a determinant of toxin activity is the presence of proteases in the midgut microenvironment of susceptible insects. Reciprocally, any alteration in the midgut protease composition of the host can result in resistance to Bt. Here in this paper, we reviewed this processes in general and presented our assays to reveal whether resistance mechanism to Bt in Diamondback Moth (DbM) larvae could be due to the function of the midgut proteases? We estimated LC50 for both probable susceptible and resistant populations in laboratory and greenhouse tests. Then, the midgut protease activities of the B. thuringiensis induced-resistant and susceptible populations of the DbM were assayed on Hemoglubin and on N-alpha-benzoyl-DL-arginine-p-nitroanilide (BapNA) for total and tryptic activities, respectively. Six hours after feeding on Bt treated and untreated canola leaves, the midguts of instar larvae of both populations were isolated. Following related protocols, peptides released through the activity of proteinases on Hemoglubin and BApNA were recorded using microplate reader. Control (Blank) was also considered with adding TCA to reaction mix before adding enzymatic extract. Data analysis indicated that there are significant differences for tryptic activity on BApNA and also for total proteolytic activity on Hemoglubin between susceptible and

  13. 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. PMID:27179865

  14. Changes in nitrogen fractions and proteolytic activities in the cotyledons of germinating lentils.

    PubMed

    Guerra, H; Nicolás, G

    1983-09-01

    Changes in ninhydrin positive material, free amino acids and protein content during germination of seeds of Lens culinaris Med. have been studied. Ninhydrin positive material and free amino acids reached their highest concentration at the fifth day of germination. Total protein which represents 21% of the total dry weight of the lentil cotyledons, suffers a degradation of only 24% in seven days of germination; in the same period of time, reserve proteins underwent a degradation of 69%, legumin being the more abundant at the start, and the more rapidly depleted. Five different classes of proteolytic activities have been reported in lentil cotyledons: caseinolytic, active against the reserve proteins of the lentil cotyledons themselves, aminopeptidase, peptidehydrolase, carboxypeptidase and dipeptidase. The removal of the axis did not seem to exert any significant influence on the enzymatic activity. PMID:6361930

  15. 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

  16. 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

  17. 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. PMID:24571675

  18. Proteolytic and Trypsin Inhibitor Activity in Germinating Jojoba Seeds (Simmondsia chinensis) 1

    PubMed Central

    Samac, Deborah; Storey, Richard

    1981-01-01

    Changes in proteolytic activity (aminopeptidase, carboxypeptidase, endopeptidase) were followed during germination (imbibition through seedling development) in extracts from cotyledons of jojoba seeds (Simmondsia chinensis). After imbibition, the cotyledons contained high levels of sulfhydryl aminopeptidase activity (APA) but low levels of serine carboxypeptidase activity (CPA). CPA increased with germination through the apparent loss of a CPA inhibitor substance in the seed. Curves showing changes in endopeptidase activity (EPA) assayed at pH 4, 5, 6, 7, and 8 during germination were distinctly different. EPA at pH 4, 5, 6, and 7 showed characteristics of sulfhydryl enzymes while activity at pH 8 was probably due to a serine type enzyme. EPA at pH 6 was inhibited early in germination by one or more substances in the seed. Activities at pH 5 and later at pH 6 were the highest of all EPA throughout germination and increases in these activities were associated with a rapid loss of protein from the cotyledons of the developing seedling. Jojoba cotyledonary extracts were found to inhibit the enzymic activity of trypsin, chymotrypsin, and pepsin but not the protease from Aspergillus saotoi. The heat-labile trypsin inhibitor substance(s) was found in commercially processed jojoba seed meal and the albumin fraction of seed proteins. Trypsin inhibitor activity decreased with germination. PMID:16662104

  19. 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

  20. Trichomonas vaginalis Cysteine Proteinases: Iron Response in Gene Expression and Proteolytic Activity.

    PubMed

    Arroyo, Rossana; Cárdenas-Guerra, Rosa Elena; Figueroa-Angulo, Elisa Elvira; Puente-Rivera, Jonathan; Zamudio-Prieto, Olga; Ortega-López, Jaime

    2015-01-01

    We focus on the iron response of Trichomonas vaginalis to gene family products such as the cysteine proteinases (CPs) involved in virulence properties. In particular, we examined the effect of iron on the gene expression regulation and function of cathepsin L-like and asparaginyl endopeptidase-like CPs as virulence factors. We addressed some important aspects about CPs genomic organization and we offer possible explanations to the fact that only few members of this large gene family are expressed at the RNA and protein levels and the way to control their proteolytic activity. We also summarized all known iron regulations of CPs at transcriptional, posttranscriptional, and posttranslational levels along with new insights into the possible epigenetic and miRNA processes. PMID:26090464

  1. MicroRNA-148b Acts as a Tumor Suppressor in Cervical Cancer by Inducing G1/S-Phase Cell Cycle Arrest and Apoptosis in a Caspase-3-Dependent Manner

    PubMed Central

    Mou, Zongmei; Xu, Xiangting; Dong, Mei; Xu, Jin

    2016-01-01

    Background The purpose of our study was to investigate the role of microRNA (miR)-148b in cervical cancer. Material/Methods The expression of miR-148b was determined in HPV-16-immortalized cervical epithelial cell line CRL-2614 cells and in cervical cancer cell line HeLa cells. The miR-148b mimics or scrambled RNA were then transfected into Hela cells. Forty-eight hours after transfection, the mRNA expression of miR-148b and DNA methyltransferase 1 (DNMT1) were confirmed. Cell proliferation ability (cell viability and colony formation ability), invasion ability, and apoptosis were assessed after transfection with miR-148b mimics or scrambled RNA, as well as the protein expression of cyclin D1 and caspase-3. Results The expression of miR-148b was significantly downregulated in HeLa cells compared with CRL2614 cells (P<0.05), but was statistically upregulated by transfection with miR-148b mimics compared with the cells transfected with scrambled RNA (P<0.05). Also, we found that the expression of DNMT1 was significantly decreased by transfection with miR-148b mimics (P<0.05). Additionally, miR-148b mimics significantly decreased the cell proliferation ability and invasion ability, and statistically induced apoptosis. Furthermore, the expression of cyclin D1 protein was significantly decreased and the expression of caspase-3 protein was significantly increased by miR-148b mimics compared with that in the cells transfected with scrambled RNA (P<0.05). Conclusions Our results suggest that overexpression of miR-148b protects against cervical cancer by inducing G1/S-phase cell cycle arrest and apoptosis through caspase-3-dependent manner, and overexpression of miR-148b might develop a therapeutic intervention for cervical cancer. PMID:27505047

  2. 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. PMID:26700145

  3. Development of a solid-phase assay for measurement of proteolytic enzyme activity

    SciTech Connect

    Varani, J.; Johnson, K.; Kaplan, J.

    1980-09-15

    A solid-phase, plate assay was developed for the measurement of proteolytic enzyme activity. In this assay procedure, radiolabeled substrates were dried onto the surface of microtiter wells. Following drying, the wells were washed two times with saline to remove the nonadherent substrate. When proteolytic enzymes were added to the wells, protein hydrolysis occurred, releasing radioactivity into the supernatant fluid. The amount of protein hydrolysis that occurred was reflected by the amount of radioactivity in the supernatant fluid. When /sup 125/I-hemoglobin was used as the substrate, it was as susceptible to hydrolysis by trypsin in the solid-phase assay as it was in solution in a standard assay procedure. Protease activity from a variety of sources (including from viable cells as well as from extracellular sources) were also able to hydrolyze the hemoglobin on the plate. /sup 125/I-Labeled serum albumen, fibrinogen, and rat pulmonary basement membrane were also susceptible to hydrolysis by trypsin in the solid phase. When (/sup 14/C)elastin was dried onto the plate, it behaved in a similar manner to elastin in solution. It was resistant to hydrolysis by nonspecific proteases such as trypsin and chymotrypsin but was highly susceptible to hydrolysis by elastase. The solid-phase plate assay has several features which recommended it for routine use. It is as sensitive as standard tube assays (and much more sensitive than routinely used colormetric assays). It is quick and convenient; there are no precipitation, centrifugation, or filtration steps. In addition, very small volumes of radioactive wastes are generated. Another advantage of the solid-phase plate assay is the resistance of the dried substrates to spontaneous breakdown and to microbial contamination. Finally, this assay is suitable for use with viable cells as well as for extracellular proteases.

  4. Role of proteolytic activation of protein kinase Cδ in the pathogenesis of prion disease

    PubMed Central

    Harischandra, Dilshan S; Kondru, Naveen; Martin, Dustin P; Kanthasamy, Arthi; Jin, Huajun; Anantharam, Vellareddy; Kanthasamy, Anumantha G

    2014-01-01

    Prion diseases are infectious and inevitably fatal neurodegenerative diseases characterized by prion replication, widespread protein aggregation and spongiform degeneration of major brain regions controlling motor function. Oxidative stress has been implicated in prion-related neuronal degeneration, but the molecular mechanisms underlying prion-induced oxidative damage are not well understood. In this study, we evaluated the role of oxidative stress-sensitive, pro-apoptotic protein kinase Cδ (PKCδ) in prion-induced neuronal cell death using cerebellar organotypic slice cultures (COSC) and mouse models of prion diseases. We found a significant upregulation of PKCδ in RML scrapie-infected COSC, as evidenced by increased levels of both PKCδ protein and its mRNA. We also found an enhanced regulatory phosphorylation of PKCδ at its two regulatory sites, Thr505 in the activation loop and Tyr311 at the caspase-3 cleavage site. The prion infection also induced proteolytic activation of PKCδ in our COSC model. Immunohistochemical analysis of scrapie-infected COSC revealed loss of PKCδ positive Purkinje cells and enhanced astrocyte proliferation. Further examination of PKCδ signaling in the RML scrapie adopted in vivo mouse model showed increased proteolytic cleavage and Tyr 311 phosphorylation of the kinase. Notably, we observed a delayed onset of scrapie-induced motor symptoms in PKCδ knockout (PKCδ−/−) mice as compared with wild-type (PKCδ+/+) mice, further substantiating the role of PKCδ in prion disease. Collectively, these data suggest that PKCδ signaling likely plays a role in the neurodegenerative processes associated with prion diseases. PMID:24576946

  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. PMID:27307585

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

    PubMed Central

    Park, Keon-Young; Li, Weiwei A.; Platt, Manu O.

    2012-01-01

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

  7. 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. PMID:25553996

  8. Effect of proteolytic activities in combination with the pectolytic activities on extractability of the fat and phenolic compounds from olives.

    PubMed

    Moustakime, Youssef; Hazzoumi, Zakaria; Amrani Joutei, Khalid

    2016-01-01

    During the extraction, a portion of oil remains trapped inside the cells and its release requires the degradation of the walls and cell membranes, especially when the fruits have not reached a maximum maturity which is likely to cause an optimal embrittlement of the parietal structures and cell membrane. This can be done by specific enzymes necessary for the degradation of various cellular barriers. Three different enzyme treatments proteolytic, pectolytic or both are applied on the Moroccan Picholine olives from veraison to maturity of the fruit. The effect of these treatments is evaluated by olive oil diffusion, its phenolic content (PC) and cellular embrittlement determination of olives during ripening. The pectolytic activities lead to a significant increase in both the oil extractability (76 % at veraison and 14 % at maturity) and the PC (up to 50 % of gain compared to the control at veraison and 27 % at maturity). The proteolytic activities applied alone have no significant effect on the extractability and the polyphenols levels of oils. Furthermore, when these proteolytic activities are added in combination with the pectolytic activities, the oil extractability is doubled at veraison and its flowing up to 99 % at maturity that barely 84 % in the control in addition to a richness of polyphenols which can reach 84 % more compared to the control. This increase in polyphenols wealth is probably due to the degradation of cell walls, cellular and vacuolar membranes by enzyme activities releasing PCs that were previously associated with these structures in the drupe. PMID:27376007

  9. Proteolytic regulation of epithelial sodium channels by urokinase plasminogen activator: cutting edge and cleavage sites.

    PubMed

    Ji, Hong-Long; Zhao, Runzhen; Komissarov, Andrey A; Chang, Yongchang; Liu, Yongfeng; Matthay, Michael A

    2015-02-27

    Plasminogen activator inhibitor 1 (PAI-1) level is extremely elevated in the edematous fluid of acutely injured lungs and pleurae. Elevated PAI-1 specifically inactivates pulmonary urokinase-type (uPA) and tissue-type plasminogen activators (tPA). We hypothesized that plasminogen activation and fibrinolysis may alter epithelial sodium channel (ENaC) activity, a key player in clearing edematous fluid. Two-chain urokinase (tcuPA) has been found to strongly stimulate heterologous human αβγ ENaC activity in a dose- and time-dependent manner. This activity of tcuPA was completely ablated by PAI-1. Furthermore, a mutation (S195A) of the active site of the enzyme also prevented ENaC activation. By comparison, three truncation mutants of the amino-terminal fragment of tcuPA still activated ENaC. uPA enzymatic activity was positively correlated with ENaC current amplitude prior to reaching the maximal level. In sharp contrast to uPA, neither single-chain tPA nor derivatives, including two-chain tPA and tenecteplase, affected ENaC activity. Furthermore, γ but not α subunit of ENaC was proteolytically cleaved at ((177)GR↓KR(180)) by tcuPA. In summary, the underlying mechanisms of urokinase-mediated activation of ENaC include release of self-inhibition, proteolysis of γ ENaC, incremental increase in opening rate, and activation of closed (electrically "silent") channels. This study for the first time demonstrates multifaceted mechanisms for uPA-mediated up-regulation of ENaC, which form the cellular and molecular rationale for the beneficial effects of urokinase in mitigating mortal pulmonary edema and pleural effusions. PMID:25555911

  10. Autosomal ichthyosis with hypotrichosis syndrome displays low matriptase proteolytic activity and is phenocopied in ST14 hypomorphic mice.

    PubMed

    List, Karin; Currie, Brooke; Scharschmidt, Tiffany C; Szabo, Roman; Shireman, Jessica; Molinolo, Alfredo; Cravatt, Benjamin F; Segre, Julia; Bugge, Thomas H

    2007-12-14

    Human autosomal recessive ichthyosis with hypotrichosis (ARIH) is an inherited disorder recently linked to homozygosity for a point mutation in the ST14 gene that causes a G827R mutation in the matriptase serine protease domain (G216 in chymotrypsin numbering). Here we show that human G827R matriptase has strongly reduced proteolytic activity toward small molecule substrates, as well as toward its candidate epidermal target, prostasin. To further investigate the possible contribution of low matriptase activity to ARIH, we generated an ST14 hypomorphic mouse strain that displays a 100-fold reduction in epidermal matriptase mRNA levels. Interestingly, unlike ST14 null mice, ST14 hypomorphic mice were viable and fertile but displayed a spectrum of abnormalities that strikingly resembled ARIH. Thus, ST14 hypomorphic mice developed hyperproliferative and retention ichthyosis with impaired desquamation, hypotrichosis with brittle, thin, uneven, and sparse hair, and tooth defects. Biochemical analysis of ST14 hypomorphic epidermis revealed reduced prostasin proteolytic activation and profilaggrin proteolytic processing, compatible with a primary role of matriptase in this process. This work strongly indicates that reduced activity of a matriptase-prostasin proteolytic cascade is the etiological origin of human ARIH and provides an important mouse model for the exploration of matriptase function in ARIH, as well as multiple other physiological and pathological processes. PMID:17940283

  11. Structural analysis and proteolytic activation of Enterococcus faecalis cytolysin, a novel lantibiotic.

    PubMed

    Booth, M C; Bogie, C P; Sahl, H G; Siezen, R J; Hatter, K L; Gilmore, M S

    1996-09-01

    Clinical isolates of Enterococcus faecalis more commonly produce a cytolysin than do commensal isolates. Epidemiologic evidence and animal-model studies have established a role for the cytolysin in the pathogenesis of enterococcal disease. The cytolysin consists of two structural subunits, CylLL and CylLS, that are activated by a third component, CylA. Genetic and biochemical characterization of CylA indicate that it is a serine protease, and that activation putatively results from cleavage of one or both cytolysin subunits. Genetic evidence also suggests that the cytolysin subunits are related to the rapidly growing class of bacteriocins termed lantibiotics. However, unlike lantibiotics, the cytolysin is lytic for eukaryotic as well as prokaryotic cells, and it consists of two structural subunits. This report describes the purification and characterization of the cytolysin subunits and detection of lanthionine-type post-translational modifications within their structures. Furthermore, the cleavage specificity of the CylA activator is reported and it is shown that proteolytic activation of both subunits is essential for activity. PMID:8898386

  12. Membrane-associated proteolytic activity in Escherichia coli that is stimulated by ATP

    SciTech Connect

    Klemes, Y.; Voellmy, R.W.; Goldberg, A.L.

    1986-05-01

    The degradation of proteins in bacteria requires metabolism energy. One important enzyme in this process is protease La, a soluble ATP-dependent protease encoded by the lon gene. However, lon mutants that lack a functional protease La still show some ATP-dependent protein breakdown. The authors have reported an ATP-stimulated endoproteolytic activity associated with the inner membrane of E. coli. This ATP-stimulated activity is found in normal levels in membranes derived from lon mutants, including strains carrying insertions in the lon gene. The membrane-bound activity hydrolyzes /sup 14/C-methylglobin at a linear rate for up to 3 hours. These fractions also contain appreciable proteolytic activity that is not affected by ATP. The stimulation by ATP requires the presence of Mg/sup 2 +/. Nonhydrolyzable ATP analogs (e.g. AMPPNP or ATP-..gamma..-S) and ADP do not enhance proteolysis. Unlike protease La, the membrane-associated enzyme does not degrade the fluorometric substrate, Glt-Ala-Ala-Phe-MNA, in an ATP-stimulated fashion, and its level is not influenced by high temperature of by the gene which regulates the heat-shock response. The enzyme is inhibited by dichloroisocoumarin and certain peptide chloromethyl ketones. They conclude that E. coli contain at least two ATP-dependent proteases with distinct specificities: one is soluble and the other is membrane-associated.

  13. 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.

  14. Caspase-8 and caspase-7 sequentially mediate proteolytic activation of acid sphingomyelinase in TNF-R1 receptosomes

    PubMed Central

    Edelmann, Bärbel; Bertsch, Uwe; Tchikov, Vladimir; Winoto-Morbach, Supandi; Perrotta, Cristiana; Jakob, Marten; Adam-Klages, Sabine; Kabelitz, Dieter; Schütze, Stefan

    2011-01-01

    We previously demonstrated that tumour necrosis factor (TNF)-induced ceramide production by endosomal acid sphingomyelinase (A-SMase) couples to apoptosis signalling via activation of cathepsin D and cleavage of Bid, resulting in caspase-9 and caspase-3 activation. The mechanism of TNF-mediated A-SMase activation within the endolysosomal compartment is poorly defined. Here, we show that TNF-induced A-SMase activation depends on functional caspase-8 and caspase-7 expression. The active forms of all three enzymes, caspase-8, caspase-7 and A-SMase, but not caspase-3, colocalize in internalized TNF receptosomes. While caspase-8 and caspase-3 are unable to induce activation of purified pro-A-SMase, we found that caspase-7 mediates A-SMase activation by direct interaction resulting in proteolytic cleavage of the 72-kDa pro-A-SMase zymogen at the non-canonical cleavage site after aspartate 253, generating an active 57 kDa A-SMase molecule. Caspase-7 down modulation revealed the functional link between caspase-7 and A-SMase, confirming proteolytic cleavage as one further mode of A-SMase activation. Our data suggest a signalling cascade within TNF receptosomes involving sequential activation of caspase-8 and caspase-7 for induction of A-SMase activation by proteolytic cleavage of pro-A-SMase. PMID:21157428

  15. 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. PMID:25376439

  16. A role for PACE4 in the proteolytic activation of anthrax toxin protective antigen.

    PubMed Central

    Gordon, V M; Rehemtulla, A; Leppla, S H

    1997-01-01

    Several bacterial protein toxins require activation by eukaryotic proteases. Previous studies have shown that anthrax toxin protective antigen (PA), Pseudomonas exotoxin A (PE), and diphtheria toxin (DT) are cleaved by furin C-terminal to the sequences RKKR, RQPR, and RVRR, respectively. Because furin-deficient cells retain some sensitivity to PA and DT, it is evident that other cellular proteases can activate these toxins. Whereas furin has been shown to require arginine residues at positions -1 and -4 for substrate recognition, another protease with an activity which could substitute for furin in toxin activation, the furin-related protease PACE4, requires basic residues in the -1, -2, and -4 positions of the substrate sequence. To examine the relative roles of furin and PACE4 in toxin activation, we used furin-deficient CHO cells (FD11 cells) transfected with either the furin (FD11/furin cells) or PACE4 (FD11/PACE4 cells) gene. Mutant PA proteins containing the cleavage sequence RAAR or KR were cytotoxic toward cells expressing only PACE4. In vitro cleavage data demonstrated that PACE4 can recognize RAAR and, to a much lesser extent, KR and RR. When extracts from PACE4-transfected cells were used as a source of proteases, PACE4 had minimal activity, indicating that it had been partially inactivated or did not remain associated with the cell membranes. Cleavage of iodinated PA containing the sequence RKKR or RAAR was detected on the surface of all cell types tested, but cleavage of a dibasic sequence was detected only intracellularly and only in cells that expressed furin or PACE4. The data provide evidence that PACE4 is present at the exterior of cells, that it plays a role in the proteolytic activation of anthrax toxin PA, and that PACE4 can activate substrates at the sequence RAAR or KR. PMID:9234799

  17. Proteolytic Activation of the Porcine Epidemic Diarrhea Coronavirus Spike Fusion Protein by Trypsin in Cell Culture

    PubMed Central

    Wicht, Oliver; Li, Wentao; Willems, Lione; Meuleman, Tom J.; Wubbolts, Richard W.; van Kuppeveld, Frank J. M.; Rottier, Peter J. M.

    2014-01-01

    ABSTRACT Isolation of porcine epidemic diarrhea coronavirus (PEDV) from clinical material in cell culture requires supplementation of trypsin. This may relate to the confinement of PEDV natural infection to the protease-rich small intestine of pigs. Our study focused on the role of protease activity on infection by investigating the spike protein of a PEDV isolate (wtPEDV) using a reverse genetics system based on the trypsin-independent cell culture-adapted strain DR13 (caPEDV). We demonstrate that trypsin acts on the wtPEDV spike protein after receptor binding. We mapped the genetic determinant for trypsin-dependent cell entry to the N-terminal region of the fusion subunit of this class I fusion protein, revealing a conserved arginine just upstream of the putative fusion peptide as the potential cleavage site. Whereas coronaviruses are typically processed by endogenous proteases of the producer or target cell, PEDV S protein activation strictly required supplementation of a protease, enabling us to study mechanistic details of proteolytic processing. IMPORTANCE Recurring PEDV epidemics constitute a serious animal health threat and an economic burden, particularly in Asia but, as of recently, also on the North-American subcontinent. Understanding the biology of PEDV is critical for combatting the infection. Here, we provide new insight into the protease-dependent cell entry of PEDV. PMID:24807723

  18. Reciprocal Degradation of YME1L and OMA1 Adapts Mitochondrial Proteolytic Activity During Stress

    PubMed Central

    Rainbolt, T. Kelly; Lebeau, Justine; Puchades, Cristina; Wiseman, R. Luke

    2016-01-01

    SUMMARY The mitochondrial inner membrane proteases YME1L and OMA1 are critical regulators of essential mitochondrial functions including inner membrane proteostasis maintenance and mitochondrial dynamics. Here, we show that YME1L and OMA1 are reciprocally degraded in response to distinct types of cellular stress. OMA1 is degraded through a YME1L-dependent mechanism in response to toxic insults that depolarize the mitochondrial membrane. Alternatively, insults that depolarize mitochondria and deplete cellular ATP stabilize active OMA1 and promote YME1L degradation. We show that the differential degradation of YME1L and OMA1 alters their proteolytic processing of the dynamin-like GTPase OPA1, a critical regulator of mitochondrial inner membrane morphology, which influences the recovery of tubular mitochondria following membrane depolarization-induced fragmentation. Our results reveal the differential stress-induced degradation of YME1L and OMA1 as a mechanism to sensitively adapt mitochondrial inner membrane protease activity and function in response to distinct types of cellular insults. PMID:26923599

  19. 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. PMID:11807127

  20. Proteolytic activation of the porcine epidemic diarrhea coronavirus spike fusion protein by trypsin in cell culture.

    PubMed

    Wicht, Oliver; Li, Wentao; Willems, Lione; Meuleman, Tom J; Wubbolts, Richard W; van Kuppeveld, Frank J M; Rottier, Peter J M; Bosch, Berend Jan

    2014-07-01

    Isolation of porcine epidemic diarrhea coronavirus (PEDV) from clinical material in cell culture requires supplementation of trypsin. This may relate to the confinement of PEDV natural infection to the protease-rich small intestine of pigs. Our study focused on the role of protease activity on infection by investigating the spike protein of a PEDV isolate (wtPEDV) using a reverse genetics system based on the trypsin-independent cell culture-adapted strain DR13 (caPEDV). We demonstrate that trypsin acts on the wtPEDV spike protein after receptor binding. We mapped the genetic determinant for trypsin-dependent cell entry to the N-terminal region of the fusion subunit of this class I fusion protein, revealing a conserved arginine just upstream of the putative fusion peptide as the potential cleavage site. Whereas coronaviruses are typically processed by endogenous proteases of the producer or target cell, PEDV S protein activation strictly required supplementation of a protease, enabling us to study mechanistic details of proteolytic processing. Importance: Recurring PEDV epidemics constitute a serious animal health threat and an economic burden, particularly in Asia but, as of recently, also on the North-American subcontinent. Understanding the biology of PEDV is critical for combatting the infection. Here, we provide new insight into the protease-dependent cell entry of PEDV. PMID:24807723

  1. Doxorubicin blocks proliferation of cancer cells through proteolytic activation of CREB3L1

    PubMed Central

    Denard, Bray; Lee, Ching; Ye, Jin

    2012-01-01

    Doxorubicin is used extensively for chemotherapy of diverse types of cancer, yet the mechanism through which it inhibits proliferation of cancer cells remains unclear. Here we report that doxorubicin stimulates de novo synthesis of ceramide, which in turn activates CREB3L1, a transcription factor synthesized as a membrane-bound precursor. Doxorubicin stimulates proteolytic cleavage of CREB3L1 by Site-1 Protease and Site-2 Protease, allowing the NH2-terminal domain of CREB3L1 to enter the nucleus where it activates transcription of genes encoding inhibitors of the cell cycle, including p21. Knockdown of CREB3L1 mRNA in human hepatoma Huh7 cells and immortalized human fibroblast SV589 cells conferred increased resistance to doxorubicin, whereas overexpression of CREB3L1 in human breast cancer MCF-7 cells markedly enhanced the sensitivity of these cells to doxorubicin. These results suggest that measurement of CREB3L1 expression may be a useful biomarker in identifying cancer cells sensitive to doxorubicin. DOI: http://dx.doi.org/10.7554/eLife.00090.001 PMID:23256041

  2. Human ZMPSTE24 disease mutations: residual proteolytic activity correlates with disease severity

    PubMed Central

    Barrowman, Jemima; Wiley, Patricia A.; Hudon-Miller, Sarah E.; Hrycyna, Christine A.; Michaelis, Susan

    2012-01-01

    The zinc metalloprotease ZMPSTE24 plays a critical role in nuclear lamin biology by cleaving the prenylated and carboxylmethylated 15-amino acid tail from the C-terminus of prelamin A to yield mature lamin A. A defect in this proteolytic event, caused by a mutation in the lamin A gene (LMNA) that eliminates the ZMPSTE24 cleavage site, underlies the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS). Likewise, mutations in the ZMPSTE24 gene that result in decreased enzyme function cause a spectrum of diseases that share certain features of premature aging. Twenty human ZMPSTE24 alleles have been identified that are associated with three disease categories of increasing severity: mandibuloacral dysplasia type B (MAD-B), severe progeria (atypical ‘HGPS’) and restrictive dermopathy (RD). To determine whether a correlation exists between decreasing ZMPSTE24 protease activity and increasing disease severity, we expressed mutant alleles of ZMPSTE24 in yeast and optimized in vivo yeast mating assays to directly compare the activity of alleles associated with each disease category. We also measured the activity of yeast crude membranes containing the ZMPSTE24 mutant proteins in vitro. We determined that, in general, the residual activity of ZMPSTE24 patient alleles correlates with disease severity. Complete loss-of-function alleles are associated with RD, whereas retention of partial, measureable activity results in MAD-B or severe progeria. Importantly, our assays can discriminate small differences in activity among the mutants, confirming that the methods presented here will be useful for characterizing any new ZMPSTE24 mutations that are discovered. PMID:22718200

  3. A psychrotrophic Burkholderia cepacia strain isolated from refrigerated raw milk showing proteolytic activity and adhesion to stainless steel.

    PubMed

    Nörnberg, Maria de Fátima Barros Leal; Mentges, Marilene Lenz; Silveira, Silvana Terra; Tondo, Eduardo César; Brandelli, Adriano

    2011-08-01

    The proteolytic activity of a psychrotrophic strain of Burkholderia cepacia isolated from refrigerated raw milk was characterized. Bur. cepacia produced proteolytic activity during growth at refrigeration temperature, with maximum activity at pH 6-7. The enzyme showed relative thermal stability in the range 40-50°C during 25 min, and maintained 80% its initial activity at 76°C/30 s. Milk coagulation assay showed that the crude protease from Bur. cepacia caused coagulation from day 2 for skimmed milk, whereas coagulation was observed from day 5 for whole milk. The adherence of this strain to stainless steel was evaluated, and the substrata had around 107 CFU/cm2 after 15 to 60 min incubation. Results on biofilm development suggest that this bacterium could adhere and to form biofilms even at refrigeration temperatures. These results indicate that Bur. cepacia may represent a potential hazardous to milk and dairy products. PMID:21457588

  4. Proteolytic activation of latent TGF-beta precedes caspase-3 activation and enhances apoptotic death of lung epithelial cells.

    PubMed

    Solovyan, Victor T; Keski-Oja, Jorma

    2006-05-01

    Transforming growth factors beta (TGF-betas) are multifunctional cytokines, which are secreted in latent forms in large latent TGF-beta complexes (LL-TGF-beta) with subsequent deposition to the extracellular matrix (ECM). While a variety of mechanisms capable of activating latent TGF-beta in vitro have been described, the physiological conditions, which promote the activation of TGF-beta in vivo are poorly understood. Mink lung epithelial cells (Mv1Lu) are a widely used model for evaluation of the effects of exogenous TGF-beta both in transcriptional and growth inhibitor assays. We find here that apoptosis of Mv1Lu cells, induced either by staurosporine or serum deprivation, is accompanied by proteolytic processing of LL-TGF-beta and the activation of endogenous TGF-beta. Activation of TGF-beta preceded caspase-3 activation and was almost completely suppressed by the serine protease inhibitor, AEBSF. Both exogenous and endogenously activated TGF-betas were able to enhance the apoptotic response of Mv1Lu cells leading to potentiation of cell death. Potentiation of cell death by activated TGF-beta was associated with downregulation of Akt and p38 MAPK, which were both activated at the initial stages of Mv1Lu apoptosis and were suppressed by exogenous TGF-beta. Pharmacological interruption of either phosphoinositide-3-kinase (PI-3K)/Akt or p38 MAPK signaling by the specific inhibitors mimicked the effect of TGF-beta leading to potentiation of cell death. Current results suggest that proteolytic activation of endogenous TGF-beta is a component of the apoptotic response, capable of modulating the death of Mv1Lu cells by inhibition of both PI-3K/Akt and p38 MAPK-dependent survival pathways. PMID:16447253

  5. Proteolytic fragmentation of inositol 1,4,5-trisphosphate receptors: a novel mechanism regulating channel activity?

    PubMed

    Wang, Liwei; Alzayady, Kamil J; Yule, David I

    2016-06-01

    Inositol 1,4,5-trisphosphate receptors (IP3 Rs) are a family of ubiquitously expressed intracellular Ca(2+) release channels. Regulation of channel activity by Ca(2+) , nucleotides, phosphorylation, protein binding partners and other cellular factors is thought to play a major role in defining the specific spatiotemporal characteristics of intracellular Ca(2+) signals. These properties are, in turn, believed pivotal for the selective and specific physiological activation of Ca(2+) -dependent effectors. IP3 Rs are also substrates for the intracellular cysteine proteases, calpain and caspase. Cleavage of the IP3 R has been proposed to play a role in apoptotic cell death by uncoupling regions important for IP3 binding from the channel domain, leaving an unregulated leaky Ca(2+) pore. Contrary to this hypothesis, we demonstrate following proteolysis that N- and C-termini of IP3 R1 remain associated, presumably through non-covalent interactions. Further, we show that complementary fragments of IP3 R1 assemble into tetrameric structures and retain their ability to be regulated robustly by IP3 . While peptide continuity is clearly not necessary for IP3 -gating of the channel, we propose that cleavage of the IP3 R peptide chain may alter other important regulatory events to modulate channel activity. In this scenario, stimulation of the cleaved IP3 R may support distinct spatiotemporal Ca(2+) signals and activation of specific effectors. Notably, in many adaptive physiological events, the non-apoptotic activities of caspase and calpain are demonstrated to be important, but the substrates of the proteases are poorly defined. We speculate that proteolytic fragmentation may represent a novel form of IP3 R regulation, which plays a role in varied adaptive physiological processes. PMID:26486785

  6. Characterization of the sites of proteolytic activation of Newcastle disease virus membrane glycoprotein precursors.

    PubMed

    Gorman, J J; Nestorowicz, A; Mitchell, S J; Corino, G L; Selleck, P W

    1988-09-01

    The F1- and F2-polypeptide components of the fusion proteins and the hemagglutinin/neuraminidase proteins of the avirulent Queensland (V4) and virulent Australia-Victoria (AuV) strains of Newcastle disease virus have been isolated and subjected to extensive primary structural analysis including amino-terminal sequence analysis and fast atom bombardment-mass spectrometry mapping. Nucleotide sequence analysis was performed on the gene which encodes the V4 hemagglutinin/neuraminidase protein. Signal peptidase cleavage was found to have occurred at the Ser31-Leu32 peptide bond of the primary translation products of the fusion protein genes. Activation cleavage of the V4 fusion protein precursor generated a sequence of -Gly-Lys-Gln-Gly84 at the carboxyl terminus of the F2-polypeptide and an amino-terminal sequence of the F1-polypeptide commencing with 86Leu-Ile-Gly-. The V4 hemagglutinin/neuraminidase protein gene was found to encode a primary translation product 45 amino acids longer at the carboxyl terminus than obtainable from the corresponding gene of the AuV strain (McGinnes, L. W., and Morrison, T. G. (1986) Virus Res. 5, 343-356). However, post-translational proteolytic processing, exclusive to the primary translation product of the V4 hemagglutinin/neuraminidase protein gene, was found to have removed the last 42 residues of this carboxyl-terminal appendage. PMID:3045120

  7. Self-assembled quantum dot-bioconjugates: characterization and use for sensing proteolytic activity

    NASA Astrophysics Data System (ADS)

    Medintz, Igor L.; Pons, Thomas; Sapsford, Kim E.; Dawson, Philip E.; Mattoussi, Hedi

    2008-04-01

    We present a characterization of the metal-affinity driven self-assembly between luminescent CdSe-ZnS core-shell semiconductor quantum dots (QDs) and either peptides or proteins appended with various length terminal polyhistidine tags. We first monitor the kinetics of self-assembly between surface-immobilized QDs and proteins/peptides under flow conditions (immobilized). To accomplish this, the QDs were immobilized onto functionalized substrates and then exposed to dye-labeled peptides/proteins. By using evanescent wave excitation of the substrate, self-assembly was assessed by monitoring the time-dependent changes in the dye fluorescence. This configuration was complemented with experiments using freely diffusing QDs and proteins/peptides (solution-phase) via energy transfer between QDs and dye-labeled proteins/peptides. Cumulatively, these measurements allowed determination of kinetic parameters, including association and dissociation rates (k on and k off) and the binding constant (K d). We find that self-assembly is rapid with an equilibrium constant K d -1 in the low nM. We next demonstrate the importance of understanding this self-assembly by creating QD-peptide bioconjugates which we employ as substrates to monitor the cleavage activity of proteolytic enzymes. This confirms that metal-affinity interactions can provide QD-bioconjugates that are functional and stable.

  8. Haemolytic and proteolytic activity of coagulase-negative staphylococci isolated from mastitis cows.

    PubMed

    Bochniarz, M; Wawron, W

    2012-01-01

    The aim of the present study was to assess the haemolytic and proteolytic activity of coagulase-negative staphylococci (CNS) isolated from cows with mastitis. The study was conducted on 100 CNS strains: S. xylosus (n=28), S. chromogenes (n=26), S. haemolyticus (n=25), S. sciuri (n=14), S. warneri (n=4), S. hominis (n=2), S. saprophyticus (n=1); 22 CNS were isolated from cows with clinical mastitis and 78 from those with subclinical mastitis. The CNS studied showed the ability to produce only alpha-haemolysin and belonged to one strain - S. haemolyticus (21.0% of isolated CNS strains). Haemolysin-positive CNS were responsible for both clinical and subclinical mastitis (22.7% and 20.5%, respectively). The ability to produce protease was found in 31.0% of CNS belonging to two strains: S. chromogenes and S. sciuri. Protease-positive CNS were the etiological factor of both clinical and subclinical mastitis (31.8% and 30.8%, respectively). All S. xylosus, S. warneri, S. hominis, and S. saprophyticus strains were found protease-negative and haemolysin-negative, irrespective of whether they caused clinical or subclinical mastitis in cows. PMID:22708359

  9. 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

  10. Urinary Proteolytic Activation of Renal Epithelial Na+ Channels in Chronic Heart Failure.

    PubMed

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

    2016-01-01

    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 with sham rats (fold increases: furin 6.7, prostasin 23.6, plasminogen 2.06, and plasmin 3.57 versus 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

  11. 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

  12. Proteolytic processing and activation of Clostridium perfringens epsilon toxin by caprine small intestinal contents.

    PubMed

    Freedman, John C; Li, Jihong; Uzal, Francisco A; McClane, Bruce A

    2014-01-01

    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. Importance: Processing and activation by intestinal proteases is a prerequisite for ETX-induced toxicity. Previous studies had characterized the activation of ETX using only arbitrarily chosen amounts of purified trypsin and/or chymotrypsin. Therefore, the current study examined ETX activation ex vivo by natural

  13. Influence of Selective Fluorination on the Biological Activity and Proteolytic Stability of Glucagon-like Peptide-1

    PubMed Central

    Meng, He; Krishnaji, Subrahmanian Tarakkad; Beinborn, Martin; Kumar, Krishna

    2009-01-01

    The relative simplicity and high specificity of peptide therapeutics has fueled recent interest. However, peptide and protein drugs generally require injection and suffer from low metabolic stability. We report here the design, synthesis and characterization of fluorinated analogues of the gut hormone peptide, GLP-1. Overall, fluorinated GLP-1 analogues displayed higher proteolytic stability with simultaneous retention of biological activity (efficacy). Fluorinated amino acids are useful for engineering peptide drug candidates and probing ligand-receptor interactions. PMID:18950150

  14. 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

  15. 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…

  16. Substrate specificity of proteolytic activity in the testes fluid and seminal plasma of the common carp Cyprinus carpio.

    PubMed

    Cejko, B I; Słowińska, M; Judycka, S; Kowalski, R K

    2016-05-01

    Substrate specificity in the seminal plasma and testes fluids of the common carp Cyprinus carpio was determined using gelatin, casein, albumin and haemoglobin. Proteolytic profiles of the testes and seminal plasma were compared. Different ranges of pH (5·5-9·5) and temperature (4-37° C) were used during incubations of seminal plasma proteinases. Differences in proteolytic activity between testes and seminal plasma may reflect specific functions of the testes and sperm ducts in semen production. Seminal plasma metalloproteinases were characterized by higher substrate specificity than were serine proteinases. Zymography optimization for seminal plasma indicated that pH 7·5 and 22° C were the optimal conditions for gel incubations. PMID:27001550

  17. 2-Photon Characterization of Optical Proteolytic Beacons for Imaging Changes in Matrix-Metalloprotease Activity in a Mouse Model of Aneurysm

    PubMed Central

    Haskett, Darren G.; Maestas, David; Howerton, Stephen J.; Smith, Tyler; Ardila, D. Catalina; Doetschman, Tom; Utzinger, Urs; McGrath, Dominic; McIntyre, J. Oliver; Vande Geest, Jonathan P.

    2016-01-01

    Abdominal aortic aneurysm is a multifactorial disease that is a leading cause of death in developed countries. Matrix-metalloproteases (MMPs) are part of the disease process, however, assessing their role in disease initiation and progression has been difficult and animal models have become essential. Combining Förster resonance energy transfer (FRET) proteolytic beacons activated in the presence of MMPs with 2-photon microscopy allows for a novel method of evaluating MMP activity within the extracellular matrix (ECM). Single and 2-photon spectra for proteolytic beacons were determined in vitro. Ex vivo experiments using the apolipoprotein E knockout angiotensin II-infused mouse model of aneurysm imaged ECM architecture simultaneously with the MMP-activated FRET beacons. 2-photon spectra of the two-color proteolytic beacons showed peaks for the individual fluorophores that enable imaging of MMP activity through proteolytic cleavage. Ex vivo imaging of the beacons within the ECM revealed both microstructure and MMP activity. 2-photon imaging of the beacons in aneurysmal tissue showed an increase in proteolytic cleavage within the ECM (p < 0.001), thus indicating an increase in MMP activity. Our data suggest that FRET-based proteolytic beacons show promise in assessing MMP activity within the ECM and will therefore allow future studies to identify the heterogeneous distribution of simultaneous ECM remodeling and protease activity in aneurysmal disease. PMID:26903264

  18. 2-Photon Characterization of Optical Proteolytic Beacons for Imaging Changes in Matrix-Metalloprotease Activity in a Mouse Model of Aneurysm.

    PubMed

    Haskett, Darren G; Maestas, David; Howerton, Stephen J; Smith, Tyler; Ardila, D Catalina; Doetschman, Tom; Utzinger, Urs; McGrath, Dominic; McIntyre, J Oliver; Vande Geest, Jonathan P

    2016-04-01

    Abdominal aortic aneurysm is a multifactorial disease that is a leading cause of death in developed countries. Matrix-metalloproteases (MMPs) are part of the disease process, however, assessing their role in disease initiation and progression has been difficult and animal models have become essential. Combining Förster resonance energy transfer (FRET) proteolytic beacons activated in the presence of MMPs with 2-photon microscopy allows for a novel method of evaluating MMP activity within the extracellular matrix (ECM). Single and 2-photon spectra for proteolytic beacons were determined in vitro. Ex vivo experiments using the apolipoprotein E knockout angiotensin II-infused mouse model of aneurysm imaged ECM architecture simultaneously with the MMP-activated FRET beacons. 2-photon spectra of the two-color proteolytic beacons showed peaks for the individual fluorophores that enable imaging of MMP activity through proteolytic cleavage. Ex vivo imaging of the beacons within the ECM revealed both microstructure and MMP activity. 2-photon imaging of the beacons in aneurysmal tissue showed an increase in proteolytic cleavage within the ECM (p<0.001), thus indicating an increase in MMP activity. Our data suggest that FRET-based proteolytic beacons show promise in assessing MMP activity within the ECM and will therefore allow future studies to identify the heterogeneous distribution of simultaneous ECM remodeling and protease activity in aneurysmal disease. PMID:26903264

  19. Polyploidisation of metastatic colon carcinoma cells by microtubule and tubulin interacting drugs: effect on proteolytic activity and invasiveness.

    PubMed

    Seiler, Nikolaus; Schneider, Yann; Gossé, Francine; Schleiffer, René; Raul, Francis

    2004-10-01

    When SW620 colon cancer-derived metastatic cells were exposed to nanomolar concentrations of Taxol, colchicine or (Z)-3,5,4'-trimethoxystilbene (R3), huge aneuploid, polynuclear cells survived the treatment. These cells released considerable amounts of the matrix metalloproteinase matrilysin (MMP-7), and tissue-type plasminogen activator (tPA) into the surrounding culture medium. MMP-7, and other proteolytic enzymes were highly expressed by these cells. In spite of their enormous size, the polyploid cells exhibited a considerable migratory capacity, as was demonstrated by their migration through an artificial basement membrane. While colchicine and R3-treated cells showed an inverse relationship between drug concentration and invasiveness, treatment with Taxol increased the capacity of the SW620 cells to penetrate through the membrane. The invasive capacity was not correlated with the induction and release of proteolytic enzymes. The idea that expression and release of proteolytic enzymes is a fundamental prerequisite of tumour cell invasiveness is generally accepted. The ability of the cells to respond to chemotactic signalling, and the filamentous structures of the cells, together with several cell adhesion factors, which are the basis of cell migration, are prerequisites of invasiveness. These factors are presumably different in the aneuploid cells produced by Taxol, colchicine and R3, and await scrutiny. PMID:15375554

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

    PubMed

    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

  1. 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

  2. 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

  3. 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.

  4. Detection of Botulinum Neurotoxin Serotype A, B, and F Proteolytic Activity in Complex Matrices with Picomolar to Femtomolar Sensitivity

    PubMed Central

    Dunning, F. Mark; Ruge, Daniel R.; Piazza, Timothy M.; Stanker, Larry H.; Zeytin, Füsûn N.

    2012-01-01

    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 precision. In this study, we present three biochemical assays for the detection and quantification of BoNT serotype A, B, and F proteolytic activities in complex matrices that offer picomolar to femtomolar sensitivity with small assay volumes and total assay times of less than 24 h. These assays consist of magnetic beads conjugated with BoNT serotype-specific antibodies that are used to purify BoNT from complex matrices before the quantification of bound BoNT proteolytic activity using the previously described BoTest reporter substrates. The matrices tested include human serum, whole milk, carrot juice, and baby food, as well as buffers containing common pharmaceutical excipients. The limits of detection were below 1 pM for BoNT/A and BoNT/F and below 10 pM for BoNT/B in most tested matrices using 200-μl samples and as low as 10 fM for BoNT/A with an increased sample volume. Together, these data describe rapid, robust, and high-throughput assays for BoNT detection that are compatible with a wide range of matrices. PMID:22923410

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

    PubMed Central

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

    2016-01-01

    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. PMID:26892926

  6. [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. PMID:27169244

  7. 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

    2016-01-01

    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. PMID:26892926

  8. 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.

  9. 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

  10. Crystallization and preliminary X-ray diffraction analysis of full-length and proteolytically activated pyruvate oxidase from Escherichia coli

    SciTech Connect

    Weidner, Annett; Neumann, Piotr; Wille, Georg; Stubbs, Milton T.; Tittmann, Kai

    2008-03-01

    The peripheral membrane flavoprotein pyruvate oxidase from E. coli has been crystallized in the full-length form and as a proteolytically activated truncation variant lacking the last 23 amino acids at the C-terminus. The thiamine diphosphate- and flavin-dependent peripheral membrane enzyme pyruvate oxidase from Escherichia coli (EcPOX) has been crystallized in the full-length form and as a proteolytically activated C-terminal truncation variant which lacks the last 23 amino acids (Δ23 EcPOX). Crystals were grown by the hanging-drop vapour-diffusion method using either protamine sulfate (full-length EcPOX) or 2-methyl-2,4-pentanediol (Δ23 EcPOX) as precipitants. Native data sets were collected at a X-ray home source to a resolution of 2.9 Å. The two forms of EcPOX crystallize in different space groups. Whereas full-length EcPOX crystallizes in the tetragonal space group P4{sub 3}2{sub 1}2 with two monomers per asymmetric unit, the crystals of Δ23 EcPOX belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1} and contain 12 monomers per asymmetric unit.

  11. Fibrinogen cleavage by the Streptococcus pyogenes extracellular cysteine protease and generation of antibodies that inhibit enzyme proteolytic activity.

    PubMed

    Matsuka, Y V; Pillai, S; Gubba, S; Musser, J M; Olmsted, S B

    1999-09-01

    The extracellular cysteine protease from Streptococcus pyogenes is a virulence factor that plays a significant role in host-pathogen interaction. Streptococcal protease is expressed as an inactive 40-kDa precursor that is autocatalytically converted into a 28-kDa mature (active) enzyme. Replacement of the single cysteine residue involved in formation of the enzyme active site with serine (C192S mutation) abolished detectable proteolytic activity and eliminated autocatalytic processing of zymogen to the mature form. In the present study, we investigated activity of the wild-type (wt) streptococcal protease toward human fibrinogen and bovine casein. The former is involved in blood coagulation, wound healing, and other aspects of hemostasis. Treatment with streptococcal protease resulted in degradation of the COOH-terminal region of fibrinogen alpha chain, indicating that fibrinogen may serve as an important substrate for this enzyme during the course of human infection. Polyclonal antibodies generated against recombinant 40- and 28-kDa (r40- and r28-kDa) forms of the C192S streptococcal protease mutant exhibited high enzyme-linked immunosorbent assay titers but demonstrated different inhibition activities toward proteolytic action of the wt enzyme. Activity of the wt protease was readily inhibited when the reaction was carried out in the presence of antibodies generated against r28-kDa C192S mutant. Antibodies produced against r40-kDa C192S mutant had no significant effect on proteolysis. These data suggest that the presence of the NH(2)-terminal prosegment prevents generation of functionally active antibodies and indicate that inhibition activity of antibodies most likely depends on their ability to bind the active-site region epitope(s) of the protein. PMID:10456870

  12. Fibrinogen Cleavage by the Streptococcus pyogenes Extracellular Cysteine Protease and Generation of Antibodies That Inhibit Enzyme Proteolytic Activity

    PubMed Central

    Matsuka, Yury V.; Pillai, Subramonia; Gubba, Siddeswar; Musser, James M.; Olmsted, Stephen B.

    1999-01-01

    The extracellular cysteine protease from Streptococcus pyogenes is a virulence factor that plays a significant role in host-pathogen interaction. Streptococcal protease is expressed as an inactive 40-kDa precursor that is autocatalytically converted into a 28-kDa mature (active) enzyme. Replacement of the single cysteine residue involved in formation of the enzyme active site with serine (C192S mutation) abolished detectable proteolytic activity and eliminated autocatalytic processing of zymogen to the mature form. In the present study, we investigated activity of the wild-type (wt) streptococcal protease toward human fibrinogen and bovine casein. The former is involved in blood coagulation, wound healing, and other aspects of hemostasis. Treatment with streptococcal protease resulted in degradation of the COOH-terminal region of fibrinogen α chain, indicating that fibrinogen may serve as an important substrate for this enzyme during the course of human infection. Polyclonal antibodies generated against recombinant 40- and 28-kDa (r40- and r28-kDa) forms of the C192S streptococcal protease mutant exhibited high enzyme-linked immunosorbent assay titers but demonstrated different inhibition activities toward proteolytic action of the wt enzyme. Activity of the wt protease was readily inhibited when the reaction was carried out in the presence of antibodies generated against r28-kDa C192S mutant. Antibodies produced against r40-kDa C192S mutant had no significant effect on proteolysis. These data suggest that the presence of the NH2-terminal prosegment prevents generation of functionally active antibodies and indicate that inhibition activity of antibodies most likely depends on their ability to bind the active-site region epitope(s) of the protein. PMID:10456870

  13. Proteolytic Activation of the Human Epithelial Sodium Channel by Trypsin IV and Trypsin I Involves Distinct Cleavage Sites*

    PubMed Central

    Haerteis, Silke; Krappitz, Annabel; Krappitz, Matteus; Murphy, Jane E.; Bertog, Marko; Krueger, Bettina; Nacken, Regina; Chung, Hyunjae; Hollenberg, Morley D.; Knecht, Wolfgang; Bunnett, Nigel W.; Korbmacher, Christoph

    2014-01-01

    Proteolytic activation is a unique feature of the epithelial sodium channel (ENaC). However, the underlying molecular mechanisms and the physiologically relevant proteases remain to be identified. The serine protease trypsin I can activate ENaC in vitro but is unlikely to be the physiologically relevant activating protease in ENaC-expressing tissues in vivo. Herein, we investigated whether human trypsin IV, a form of trypsin that is co-expressed in several extrapancreatic epithelial cells with ENaC, can activate human ENaC. In Xenopus laevis oocytes, we monitored proteolytic activation of ENaC currents and the appearance of γENaC cleavage products at the cell surface. We demonstrated that trypsin IV and trypsin I can stimulate ENaC heterologously expressed in oocytes. ENaC cleavage and activation by trypsin IV but not by trypsin I required a critical cleavage site (Lys-189) in the extracellular domain of the γ-subunit. In contrast, channel activation by trypsin I was prevented by mutating three putative cleavage sites (Lys-168, Lys-170, and Arg-172) in addition to mutating previously described prostasin (RKRK178), plasmin (Lys-189), and neutrophil elastase (Val-182 and Val-193) sites. Moreover, we found that trypsin IV is expressed in human renal epithelial cells and can increase ENaC-mediated sodium transport in cultured human airway epithelial cells. Thus, trypsin IV may regulate ENaC function in epithelial tissues. Our results show, for the first time, that trypsin IV can stimulate ENaC and that trypsin IV and trypsin I activate ENaC by cleavage at distinct sites. The presence of distinct cleavage sites may be important for ENaC regulation by tissue-specific proteases. PMID:24841206

  14. 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

  15. 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

  16. A comparison of endogenous and microbial proteolytic activities during fast fermentation of silver carp inoculated with Lactobacillus plantarum.

    PubMed

    Yang, Fang; Xia, Wen-Shui; Zhang, Xiao-Wei; Xu, Yan-Shun; Jiang, Qi-Xing

    2016-09-15

    The study was aimed to investigate different roles of endogenous and Lactobacillus plantarum proteases during fast fermentation of silver carp. The results show that endogenous proteases could degrade both sarcoplasmic and myofibrillar proteins. In contrast, L. plantarum had low proteinase activities and could only hydrolyze sarcoplasmic peptides. This indicates that gel properties could be mainly affected by endogenous proteolysis while microbial proteolysis contributed to the production of smaller peptides and free amino acids which may be related to flavor and taste. Texture and free amino acid analyses verified these hypotheses. It shows that endogenous lysosomal proteases were the major contributors for the decrease of gel strength while L. plantarum proteolytic activities could lead to the increase of aspartic acid, glutamic acid, and alanine, which may result in umami and sweet taste; and also lead to a rise in some amino acids which were volatile compounds precursors. PMID:27080883

  17. Increased proteolytic processing of full-length Gli2 transcription factor reduces the Hedgehog pathway activity in vivo

    PubMed Central

    Li, Juan; Wang, Chengbing; Pan, Yong; Bai, Zengliang; Wang, Baolin

    2011-01-01

    The proteolytic processing of Gli2 and Gli3 full-length transcription factors into repressors is a key step of the regulation in Hedgehog (Hh) signaling. The differential Gli2 and Gli3 processing is controlled by the processing determinant domain or PDD, but its significance is not clear. We generated a Gli2 mutant allele, Gli23PDD, in which the Gli3PDD substitutes for the Gli2PDD. As expected, Gli23PDD is processed more efficiently and at the different position as compared to Gli2, indicating that PDD also determines the extent and site of Gli2 and Gli3 processing in vivo. The increase in levels of the Gli2 repressor in Gli23PDD mutant reduces the Hh pathway activity. Gli23PDD processing is still regulated by Hh signaling. These results indicate that the proper balance between the Gli2 full-length activator and repressor is essential for Hh signaling. PMID:21337666

  18. Defect of vacuolar protein sorting stimulates proteolytic processing of human urokinase-type plasminogen activator in the yeast Hansenula polymorpha.

    PubMed

    Agaphonov, Michael; Romanova, Nina; Sokolov, Sviatoslav; Iline, Anna; Kalebina, Tatyana; Gellissen, Gerd; Ter-Avanesyan, Michael

    2005-11-01

    Human urokinase-type plasminogen activator (uPA) is poorly secreted by yeast cells. Here, we have selected Hansenula polymorpha mutants with increased productivity of active extracellular uPA. Several of the obtained mutants also demonstrated a defect of sorting of carboxypeptidase Y to the vacuole and the mutant loci have been identified in six of them. All these mutations damaged genes involved in protein traffic between the Golgi apparatus and the vacuole, namely PEP3, VPS8, VPS10, VPS17, and VPS35. We have shown that inactivation of the VPS10 gene encoding the vacuolar protein sorting receptor does not increase uPA secretion but stimulates its proteolytic processing. PMID:16181812

  19. Cleavage of Chordin by Xolloid Metalloprotease Suggests a Role for Proteolytic Processing in the Regulation of Spemann Organizer Activity

    PubMed Central

    Piccolo, Stefano; Agius, Eric; Lu, Bin; Goodman, Shelley; Dale, Leslie

    2011-01-01

    Summary The Xolloid secreted metalloprotease, a tolloid-related protein, was found to cleave Chordin and Chordin/BMP-4 complexes at two specific sites in biochemical experiments. Xolloid mRNA blocks secondary axes caused by chordin, but not by noggin, follistatin, or dominant-negative BMP receptor, mRNA injection. Xolloid-treated Chordin protein was unable to antagonize BMP activity. Furthermore, Xolloid digestion released biologically active BMPs from Chordin/BMP inactive complexes. Injection of dominant-negative Xolloid mRNA indicated that the in vivo function of Xolloid is to limit the extent of Spemann’s organizer field. We propose that Xolloid regulates organizer function by a novel proteolytic mechanism involving a double inhibition pathway required to pattern the dorsoventral axis: XOLL⊣CHD⊣BMPs→BMPR PMID:9363949

  20. Analysis of cathepsin and furin proteolytic enzymes involved in viral fusion protein activation in cells of the bat reservoir host.

    PubMed

    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

  1. 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

  2. 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. PMID:26608755

  3. Plasminogen-dependent and -independent proteolytic activity of murine endothelioma cells with targeted inactivation of fibrinolytic genes.

    PubMed

    Lijnen, H R; Wagner, E F; Collen, D

    1997-02-01

    Plasminogen-dependent and -independent proteolytic activity of marine endothelioma (End) cells that were derived from mice with targeted inactivation of the tissue-type plasminogen activator (t-PA-/-), urokinase-type plasminogen activator (u-PA-/-) or plasminogen activator inhibitor-1 (PAI-1-/-) genes was studied with the use of fibrin and extracellular matrix degradation assays. In a buffer milieu, the activation rate of plasminogen (final concentration 0.25 microM) with wild-type and t-PA-/- End cells (3 x 10(4) to 4 x 10(6) cells/ml) was comparable, but it was about 4-fold reduced with u-PA-/- End cells and 3-fold enhanced with PAI-1-/- End cells. Plasminogen activation was markedly reduced by addition of amiloride or of anti-murine u-PA antibodies but not by addition of anti-murine t-PA antibodies, and it was not stimulated by addition of fibrin. Lysis of 125I-fibrin labeled matrix in the presence of plasminogen was comparable with wild-type, t-PA-/- and PAI-1-/- End cells (50% lysis in 3 h with 0.7 to 1.5 x 10(6) cells/ml), but was significantly reduced with u-PA-/- End cells (50% lysis in 20 h with 0.87 x 10(6) cells/ml). Lysis of 3H-proline labeled extracellular matrix in the presence of plasminogen with wild-type, t-PA-/- and PAI-1-/- End cells (20% lysis in 48 h with 3 to 5 x 10(6) cells/ml) was comparable, but it was virtually abolished with u-PA-/- End cells. In the absence of plasminogen, lysis of both the fibrin and the extracellular matrix by all four cell types was drastically reduced and was virtually abolished by addition of phenylmethylsulfonylfluoride or 1,10 phenanthroline. These data indicate that the proteolytic activity of the transformed murine endothelioma cells, measured in plasminogen activation or matrix degradation assays, is essentially u-PA-related and largely plasminogen-dependent. PMID:9157597

  4. Effects of prunetin on the proteolytic activity, secretion and gene expression of MMP-3 in vitro and production of MMP-3 in vivo.

    PubMed

    Nam, Dae Cheol; Kim, Bo Kun; Lee, Hyun Jae; Shin, Hyun-Dae; Lee, Choong Jae; Hwang, Sun-Chul

    2016-03-01

    We investigated whether prunetin affects the proteolytic activity, secretion, and gene expression 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 eff ect of prunetin. Rabbit articular chondrocytes were cultured in a monolayer, and reverse transcription-polymerase chain reaction (RT-PCR) was used to measure interleukin-1β (IL-1β)-induced expression of MMP-3, MMP-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), and ADAMTS-5. In rabbit articular chondrocytes, the effects of prunetin on IL-1β-induced secretion and proteolytic activity of MMP-3 were investigated using western blot analysis and casein zymography, respectively. The eff ect of prunetin on MMP-3 protein production was also examined in vivo. The results were as follows: (1) prunetin inhibited the gene expression of MMP-3, MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5; (2) prunetin inhibited the secretion and proteolytic activity of MMP-3; (3) prunetin suppressed the production of MMP-3 protein in vivo. These results suggest that prunetin can regulate the gene expression, secretion, and proteolytic activity of MMP-3, by directly acting on articular chondrocytes. PMID:26937219

  5. Effects of prunetin on the proteolytic activity, secretion and gene expression of MMP-3 in vitro and production of MMP-3 in vivo

    PubMed Central

    Nam, Dae Cheol; Kim, Bo Kun; Lee, Hyun Jae; Shin, Hyun-Dae

    2016-01-01

    We investigated whether prunetin affects the proteolytic activity, secretion, and gene expression 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 eff ect of prunetin. Rabbit articular chondrocytes were cultured in a monolayer, and reverse transcription-polymerase chain reaction (RT-PCR) was used to measure interleukin-1β (IL-1β)-induced expression of MMP-3, MMP-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), and ADAMTS-5. In rabbit articular chondrocytes, the effects of prunetin on IL-1β-induced secretion and proteolytic activity of MMP-3 were investigated using western blot analysis and casein zymography, respectively. The eff ect of prunetin on MMP-3 protein production was also examined in vivo. The results were as follows: (1) prunetin inhibited the gene expression of MMP-3, MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5; (2) prunetin inhibited the secretion and proteolytic activity of MMP-3; (3) prunetin suppressed the production of MMP-3 protein in vivo. These results suggest that prunetin can regulate the gene expression, secretion, and proteolytic activity of MMP-3, by directly acting on articular chondrocytes. PMID:26937219

  6. Increased mRNA levels for components of the lysosomal, Ca2+-activated, and ATP-ubiquitin-dependent proteolytic pathways in skeletal muscle from head trauma patients.

    PubMed Central

    Mansoor, O; Beaufrere, B; Boirie, Y; Ralliere, C; Taillandier, D; Aurousseau, E; Schoeffler, P; Arnal, M; Attaix, D

    1996-01-01

    The cellular mechanisms responsible for enhanced muscle protein breakdown in hospitalized patients, which frequently results in lean body wasting, are unknown. To determine whether the lysosomal, Ca2+-activated, and ubiquitin-proteasome proteolytic pathways are activated, we measured mRNA levels for components of these processes in muscle biopsies from severe head trauma patients. These patients exhibited negative nitrogen balance and increased rates of whole-body protein breakdown (assessed by [13C]leucine infusion) and of myofibrillar protein breakdown (assessed by 3-methylhistidine urinary excretion). Increased muscle mRNA levels for cathepsin D, m-calpain, and critical components of the ubiquitin proteolytic pathway (i.e., ubiquitin, the 14-kDa ubiquitin-conjugating enzyme E2, and proteasome subunits) paralleled these metabolic adaptations. The data clearly support a role for multiple proteolytic processes in increased muscle proteolysis. The ubiquitin proteolytic pathway could be activated by altered glucocorticoid production and/or increased circulating levels of interleukin 1beta and interleukin 6 observed in head trauma patients and account for the breakdown of myofibrillar proteins, as was recently reported in animal studies. Images Fig. 1 Fig. 1 Fig. 3 Fig. 4 PMID:8610106

  7. 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

  8. 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. PMID:26877164

  9. Piperine Induces Hepatic Low-Density Lipoprotein Receptor Expression through Proteolytic Activation of Sterol Regulatory Element-Binding Proteins

    PubMed Central

    Ochiai, Ayasa; Miyata, Shingo; Shimizu, Makoto; Inoue, Jun; Sato, Ryuichiro

    2015-01-01

    Elevated plasma low-density lipoprotein (LDL) cholesterol is considered as a risk factor for atherosclerosis. Because the hepatic LDL receptor (LDLR) uptakes plasma lipoproteins and lowers plasma LDL cholesterol, the activation of LDLR is a promising drug target for atherosclerosis. In the present study, we identified the naturally occurring alkaloid piperine, as an inducer of LDLR gene expression by screening the effectors of human LDLR promoter. The treatment of HepG2 cells with piperine increased LDLR expression at mRNA and protein levels and stimulated LDL uptake. Subsequent luciferase reporter gene assays revealed that the mutation of sterol regulatory element-binding protein (SREBP)-binding element abolished the piperine-mediated induction of LDLR promoter activity. Further, piperine treatments increased mRNA levels of several SREBP targets and mature forms of SREBPs. However, the piperine-mediated induction of the mature forms of SREBPs was not observed in SRD–15 cells, which lack insulin-induced gene–1 (Insig–1) and Insig–2. Finally, the knockdown of SREBPs completely abolished the piperine-meditated induction of LDLR gene expression in HepG2 cells, indicating that piperine stimulates the proteolytic activation of SREBP and subsequent induction of LDLR expression and activity. PMID:26431033

  10. Purification and characterization of a proteolytic active fragment of DNA topoisomerase I from the brine shrimp Artemia franciscana (Crustacea Anostraca).

    PubMed Central

    Badaracco, G; Landsberger, N; Benfante, R

    1992-01-01

    The ATP-independent type I topoisomerase from the crustacean Artemia franciscana was purified to near-homogeneity. Its activity was measured by an assay that uses the formation of an enzyme-cleaved DNA complex in the presence of the specific inhibitor camptothecin. The purification procedure is reported. Purified topoisomerase is a single-subunit enzyme with a molecular mass of 63 kDa. Immunoblot performed on the different steps of purification shows that the purified 63 kDa peptide is a proteolytic fragment of a protein with a molecular mass of 110 kDa. Similarly to the other purified eukaryotic topoisomerases, the crustacean enzyme does not require a bivalent cation for activity, but is stimulated in the presence of 10 mM-MgCl2; moreover, it can relax both negative and positive superhelical turns. The enzyme activity is strongly inhibited by the antitumour drug camptothecin. The enzyme inhibition is related to the stabilization of the cleavable complex between topoisomerase I and DNA. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:1311554

  11. 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

  12. Proteolytic activation cascade of the Netherton syndrome-defective protein, LEKTI, in the epidermis: implications for skin homeostasis.

    PubMed

    Fortugno, Paola; Bresciani, Alberto; Paolini, Chantal; Pazzagli, Chiara; El Hachem, May; D'Alessio, Marina; Zambruno, Giovanna

    2011-11-01

    Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is the defective protein of the ichthyosiform condition Netherton syndrome (NS). Strongly expressed in the most differentiated epidermal layers, LEKTI is a serine protease inhibitor synthesized as three different high-molecular-weight precursors, which are rapidly processed into shorter fragments and secreted extracellularly. LEKTI polypeptides interact with several proteases to regulate skin barrier homeostasis as well as inflammatory and/or immunoallergic responses. Here, by combining antibody mapping, N-terminal sequencing, and site-specific mutagenesis, we defined the amino-acid sequence of most of the LEKTI polypeptides physiologically generated in human epidermis. We also identified three processing intermediates not described so far. Hence, a proteolytic cascade model for LEKTI activation is proposed. We then pinpointed the most effective fragments against the desquamation-related kallikreins (KLKs) and we proved that LEKTI is involved in stratum corneum shedding as some of its polypeptides inhibit the KLK-mediated proteolysis of desmoglein-1. Finally, we quantified the individual LEKTI fragments in the uppermost epidermis, showing that the ratios between LEKTI polypeptides and active KLK5 are compatible with a fine-tuned inhibition. These findings are relevant both to the understanding of skin homeostasis regulation and to the design of novel therapeutic strategies for NS. PMID:21697885

  13. The fungal metabolite gliotoxin inhibits proteasome proteolytic activity and induces an irreversible pseudocystic transformation and cell death in Tritrichomonas foetus.

    PubMed

    Pereira-Neves, Antonio; Menna-Barreto, Rubem F S; Benchimol, Marlene

    2016-08-01

    Proteasomal proteolysis is required for a wide range of cellular processes, including protein quality control, cell cycle progression, cell death and metabolic adaptation to environment changes or stress responses. Proteasome inhibitors are useful compounds for determining the roles of proteasome in eukaryotic cells. Here, we investigated the effects of gliotoxin, a proteasome inhibitor, on the cell growth, replication, ultrastructure, DNA integrity and proteasomal proteolytic activity of the protist parasite Tritrichomonas foetus. The effect of gliotoxin on the transformation of T. foetus to endoflagellar form (EFF), also known as pseudocyst, was investigated. Gliotoxin inhibited the culture growth, arrested cell cycle, and provoked a trichomonacidal effect in a dose-dependent manner. Parasites treated with gliotoxin displayed features typical of cell death, such as membrane blebbing, concentric membrane whorls containing remnants of organelles, intense cytosolic and nuclear vacuolisation, chromatin condensation, DNA fragmentation, cytoplasmic disintegration and plasma membrane disruption. The proteasomal peptidase activity was inhibited by gliotoxin in a dose-dependent manner. Gliotoxin treatment also induced an irreversible EFF transformation in a dose/time-dependent manner. We compared morphological characteristics between gliotoxin- and cold-induced EFF parasites. Our results suggest that gliotoxin could induce EFF transformation by a mechanism distinct from that provoked by cold temperature. This study further contributes to a better understanding of the role of proteasome system in cell cycle, cell death and EFF transformation in T. foetus. PMID:27106236

  14. 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

  15. C-Terminal extension of a plant cysteine protease modulates proteolytic activity through a partial inhibitory mechanism.

    PubMed

    Dutta, Sruti; Choudhury, Debi; Dattagupta, Jiban K; Biswas, Sampa

    2011-09-01

    The amino acid sequence of ervatamin-C, a thermostable cysteine protease from a tropical plant, revealed an additional 24-amino-acid extension at its C-terminus (CT). The role of this extension peptide in zymogen activation, catalytic activity, folding and stability of the protease is reported. For this study, we expressed two recombinant forms of the protease in Escherichia coli, one retaining the CT-extension and the other with it truncated. The enzyme with the extension shows autocatalytic zymogen activation at a higher pH of 8.0, whereas deletion of the extension results in a more active form of the enzyme. This CT-extension was not found to be cleaved during autocatalysis or by limited proteolysis by different external proteases. Molecular modeling and simulation studies revealed that the CT-extension blocks some of the substrate-binding unprimed subsites including the specificity-determining subsite (S2) of the enzyme and thereby partially occludes accessibility of the substrates to the active site, which also corroborates the experimental observations. The CT-extension in the model structure shows tight packing with the catalytic domain of the enzyme, mediated by strong hydrophobic and H-bond interactions, thus restricting accessibility of its cleavage sites to the protease itself or to the external proteases. Kinetic stability analyses (T(50) and t(1/2) ) and refolding experiments show similar thermal stability and refolding efficiency for both forms. These data suggest that the CT-extension has an inhibitory role in the proteolytic activity of ervatamin-C but does not have a major role either in stabilizing the enzyme or in its folding mechanism. PMID:21707922

  16. Proteolytic activity of the purified hormone-binding subunit in the estrogen receptor.

    PubMed Central

    Molinari, A M; Abbondanza, C; Armetta, I; Medici, N; Minucci, S; Moncharmont, B; Nigro, V; Puca, G A

    1991-01-01

    The hormone-binding subunit of the calf uterus estradiol receptor was purified as a hormone-free molecule. Immunoaffinity chromatography with a specific monoclonal antibody was used as the final step. The purified subunit was specifically labeled by radioactive diisopropyl fluorophosphate. The diisopropyl fluorophosphate-labeled amino acid was serine. The purified receptor was able to release the fluorogenic or chromogenic group from synthetic peptides containing phenylalanine at the carboxyl terminus. This occurred only in the presence of estradiol and was hampered by aprotinin and diisopropyl fluorophosphate. Estradiol-dependent hydrolytic activity was also found in the eluate from gel slices after SDS/PAGE of purified receptor. This activity comigrated with the renaturable estradiol-binding activity. The estradiol antagonists 4-hydroxytamoxifen and ICI 164,384 as well as other steroid hormones were unable to activate this hydrolytic activity. Images PMID:1709742

  17. Proteolytic activity of the purified hormone-binding subunit in the estrogen receptor.

    PubMed

    Molinari, A M; Abbondanza, C; Armetta, I; Medici, N; Minucci, S; Moncharmont, B; Nigro, V; Puca, G A

    1991-05-15

    The hormone-binding subunit of the calf uterus estradiol receptor was purified as a hormone-free molecule. Immunoaffinity chromatography with a specific monoclonal antibody was used as the final step. The purified subunit was specifically labeled by radioactive diisopropyl fluorophosphate. The diisopropyl fluorophosphate-labeled amino acid was serine. The purified receptor was able to release the fluorogenic or chromogenic group from synthetic peptides containing phenylalanine at the carboxyl terminus. This occurred only in the presence of estradiol and was hampered by aprotinin and diisopropyl fluorophosphate. Estradiol-dependent hydrolytic activity was also found in the eluate from gel slices after SDS/PAGE of purified receptor. This activity comigrated with the renaturable estradiol-binding activity. The estradiol antagonists 4-hydroxytamoxifen and ICI 164,384 as well as other steroid hormones were unable to activate this hydrolytic activity. PMID:1709742

  18. Lactoferrin Is an Allosteric Enhancer of the Proteolytic Activity of Cathepsin G

    PubMed Central

    Eipper, Steffen; Steiner, Robin; Lesner, Adam; Sienczyk, Marcin; Palesch, David; Halatsch, Marc-Eric; Zaczynska, Ewa; Heim, Christopher; Hartmann, Marcus D.; Zimecki, Michal; Wirtz, Christian Rainer; Burster, Timo

    2016-01-01

    Protease-mediated degradation of proteins is critical in a plethora of physiological processes. Neutrophils secrete serine proteases including cathepsin G (CatG), neutrophile elastase (NE), and proteinase 3 (PR3) together with lactoferrin (LF) as a first cellular immune response against pathogens. Here, we demonstrate that LF increases the catalytic activity of CatG at physiological concentration, with its highest enhancing capacity under acidic (pH 5.0) conditions, and broadens the substrate selectivity of CatG. On a functional level, the enzymatic activity of CatG was increased in the presence of LF in granulocyte-derived supernatant. Furthermore, LF enhanced CatG-induced activation of platelets as determined by cell surface expression of CD62P. Consequently, LF-mediated enhancement of CatG activity might promote innate immunity during acute inflammation. PMID:26986619

  19. Activity of Proteolytic Enzymes and Level of Cystatin C in the Peripartum Period.

    PubMed

    Cyganek, Anna; Wyczalkowska-Tomasik, Aleksandra; Jarmuzek, Patrycja; Grzechocinska, Barbara; Jabiry-Zieniewicz, Zoulikha; Paczek, Leszek; Wielgos, Miroslaw

    2016-01-01

    Objectives. The aim of the study was to evaluate the activity of cathepsin B, collagenases, trypsin, and plasmin and concentration of cystatin C in serum of healthy pregnant women in peripartum period. Study Design. The study group included 45 women in uncomplicated pregnancies. Blood samples were collected in four time points. Enzyme activity was measured by spectrofluorometric method. The level of cystatin C was measured using immunonephelometric method. Results. Mean activity of cathepsin B and the level of serum cystatin C were significantly higher in the study group. Collagenase activity was significantly lower in the study group than the control group. No differences in collagenase, plasmin, and trypsin activity on each day of the peripartum period were found. Conclusion. High activity of cathepsin B and increased level of cystatin C are typical for women in late pregnancy. Those levels significantly decrease after delivery which can be associated with potential role of those markers in placental separation. The insignificant changes of cystatin C level in the peripartum period seem to exclude the possibility of using cystatin C as a marker for renal insufficiency in the peripartum period but additional research is necessary to investigate the matter further. PMID:26904684

  20. Activity of Proteolytic Enzymes and Level of Cystatin C in the Peripartum Period

    PubMed Central

    Cyganek, Anna; Wyczalkowska-Tomasik, Aleksandra; Jarmuzek, Patrycja; Grzechocinska, Barbara; Jabiry-Zieniewicz, Zoulikha; Paczek, Leszek; Wielgos, Miroslaw

    2016-01-01

    Objectives. The aim of the study was to evaluate the activity of cathepsin B, collagenases, trypsin, and plasmin and concentration of cystatin C in serum of healthy pregnant women in peripartum period. Study Design. The study group included 45 women in uncomplicated pregnancies. Blood samples were collected in four time points. Enzyme activity was measured by spectrofluorometric method. The level of cystatin C was measured using immunonephelometric method. Results. Mean activity of cathepsin B and the level of serum cystatin C were significantly higher in the study group. Collagenase activity was significantly lower in the study group than the control group. No differences in collagenase, plasmin, and trypsin activity on each day of the peripartum period were found. Conclusion. High activity of cathepsin B and increased level of cystatin C are typical for women in late pregnancy. Those levels significantly decrease after delivery which can be associated with potential role of those markers in placental separation. The insignificant changes of cystatin C level in the peripartum period seem to exclude the possibility of using cystatin C as a marker for renal insufficiency in the peripartum period but additional research is necessary to investigate the matter further. PMID:26904684

  1. The 420K LEKTI variant alters LEKTI proteolytic activation and results in protease deregulation: implications for atopic dermatitis.

    PubMed

    Fortugno, Paola; Furio, Laetitia; Teson, Massimo; Berretti, Matteo; El Hachem, May; Zambruno, Giovanna; Hovnanian, Alain; D'Alessio, Marina

    2012-10-01

    Lymphoepithelial Kazal-type related inhibitor (LEKTI) is a multidomain serine protease inhibitor which plays a central role in skin permeability barrier and allergy. Loss-of-function mutations in the LEKTI encoding gene SPINK5 cause Netherton syndrome, a rare and severe genetic skin disease with a profound skin barrier defect and atopic manifestations. Several studies also reported genetic association between the multifactorial disease atopic dermatitis (AD) and a frequent and non-conservative LEKTI variant, E420K, in different populations. Here, we provide evidence that the 420K variant impacts on LEKTI function by increasing the likelihood of furin-dependent LEKTI precursor cleavage within the linker region D6-D7. This results in the reversal of the cleavage priorities for LEKTI proteolytic activation and prevents the formation of the LEKTI fragment D6D9 known to display the strongest inhibitory activity against kallikrein (KLK) 5-mediated desmoglein-1 (DSG1) degradation. Using in situ and gel zymographies, we show that the modification of the subtle balance in LEKTI inhibitory fragments leads to enhanced KLK5, KLK7 and elastase-2 (ELA-2) activities in 420KK epidermis. By immunohistochemistry and western blot analyses, we found that increased epidermal protease activity correlates with reduced DSG1 protein expression and accelerated profilaggrin proteolysis. All changes determined by the presence of residue 420K within the LEKTI sequence likely contribute to defective skin barrier permeability. Remarkably, LEKTI 420KK epidermis displays an increased expression of the proallergic cytokine thymic stromal lymphopoietin (TSLP). This is the first functional evidence supporting association studies which identified the 420K LEKTI variant as a predisposing factor to AD, in combination with other genetic and environmental factors. PMID:22730493

  2. Selective photodetection and photodynamic therapy for prostate cancer through targeting of proteolytic activity.

    PubMed

    Zuluaga, Maria-Fernanda; Sekkat, Nawal; Gabriel, Doris; van den Bergh, Hubert; Lange, Norbert

    2013-03-01

    Frequent side effects of radical treatment modalities and the availability of novel diagnostics have raised the interest in focal therapies for localized prostate cancer. To improve the selectivity and therapeutic efficacy of such therapies, we developed a minimally invasive procedure based on a novel polymeric photosensitizer prodrug sensitive to urokinase-type plasminogen activator (uPA). The compound is inactive in its prodrug form and accumulates passively at the tumor site by the enhanced permeability and retention effect. There, the prodrug is selectively converted to its photoactive form by uPA, which is overexpressed by prostate cancer cells. Irradiation of the activated photosensitizer exerts a tumor-selective phototoxic effect. The prodrug alone (8 μmol/L) showed no toxic effect on PC-3 cells, but upon irradiation the cell viability was reduced by 90%. In vivo, after systemic administration of the prodrug, PC-3 xenografts became selectively fluorescent. This is indicative of the prodrug accumulation in the tumor and selective local enzymatic activation. Qualitative analysis of the activated compound confirmed that the enzymatic cleavage occurred selectively in the tumor, with only trace amounts in the neighboring skin or muscle. Subsequent photodynamic therapy studies showed complete tumor eradication of animals treated with light (150 J/cm(2) at 665 nm) 16 hours after the injection of the prodrug (7.5 mg/kg). These promising results evidence the excellent selectivity of our prodrug with the potential to be used for both imaging and therapy for localized prostate cancer. PMID:23270928

  3. 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

  4. 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

  5. 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

  6. Effect of fluticasone propionate on neutrophil chemotaxis, superoxide generation, and extracellular proteolytic activity in vitro.

    PubMed Central

    Llewellyn-Jones, C. G.; Hill, S. L.; Stockley, R. A.

    1994-01-01

    BACKGROUND--Corticosteroids are widely used in the treatment of many inflammatory conditions but the exact mode of action on neutrophil function is uncertain. Fluticasone propionate is a new topically active synthetic steroid which can be measured in body fluids and which undergoes first pass metabolism. METHODS--The effects of fluticasone propionate on the function of neutrophils isolated from normal, healthy control subjects and on the chemotactic activity of sputum sol phase were assessed. RESULTS--Preincubation of neutrophils with fluticasone propionate reduced the chemotactic response to 10(-8) mol/l F-Met-Leu-Phe (FMLP) and to a 1:5 dilution of sputum sol phase in a dose dependent manner. Furthermore, when fluticasone propionate was added to sputum from eight patients with stable chronic obstructive bronchitis the chemotactic activity of a 1:5 dilution of the sol phase fell from a mean (SE) value of 22.2 (1.21) cells/field to 19.6 (0.89), 17.1 (0.74), and 11.9 (0.6) cells field at 1 mumol/l, 10 mumol/l, and 100 mumol/l, respectively. In further experiments fluticasone propionate preincubated with neutrophils inhibited fibronectin degradation by resting cells and by cells stimulated by FMLP (15.2% inhibition of resting cells, 5.1% inhibition of stimulated cells with 1 mumol/l fluticasone propionate, 24% and 18.7% inhibition respectively at 100 mumol/l fluticasone propionate. Fluticasone propionate had no effect on generation of superoxide anion by resting or stimulated cells. CONCLUSIONS--These results indicate that fluticasone propionate has a direct suppressive effect on several aspects of neutrophil function and may suggest a role for this agent in the modulation of neutrophil mediated damage to connective tissue. PMID:8202875

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

    PubMed

    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

  8. Identification of the N-glycosylation sites on glutamate carboxypeptidase II necessary for proteolytic activity.

    PubMed

    Barinka, Cyril; Sácha, Pavel; Sklenár, Jan; Man, Petr; Bezouska, Karel; Slusher, Barbara S; Konvalinka, Jan

    2004-06-01

    Glutamate carboxypeptidase II (GCPII) is a membrane peptidase expressed in the prostate, central and peripheral nervous system, kidney, small intestine, and tumor-associated neovasculature. The GCPII form expressed in the central nervous system, termed NAALADase, is responsible for the cleavage of N-acetyl-L-aspartyl-L-glutamate (NAAG) yielding free glutamate in the synaptic cleft, and is implicated in various pathologic conditions associated with glutamate excitotoxicity. The prostate form of GCPII, termed prostate-specific membrane antigen (PSMA), is up-regulated in cancer and used as an effective prostate cancer marker. Little is known about the structure of this important pharmaceutical target. As a type II membrane protein, GCPII is heavily glycosylated. In this paper we show that N-glycosylation is vital for proper folding and subsequent secretion of human GCPII. Analysis of the predicted N-glycosylation sites also provides evidence that these sites are critical for GCPII carboxypeptidase activity. We confirm that all predicted N-glycosylation sites are occupied by an oligosaccharide moiety and show that glycosylation at sites distant from the putative catalytic domain is critical for the NAAG-hydrolyzing activity of GCPII calling the validity of previously described structural models of GCPII into question. PMID:15152093

  9. 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

  10. Proteolytic activity of Escherichia coli oligopeptidase B against proline-rich antimicrobial peptides.

    PubMed

    Mattiuzzo, Maura; De Gobba, Cristian; Runti, Giulia; Mardirossian, Mario; Bandiera, Antonella; Gennaro, Renato; Scocchi, Marco

    2014-02-28

    Oligopeptidase B (OpdB) is a serine peptidase widespread among bacteria and protozoa that has emerged as a virulence factor despite its function has not yet been precisely established. By using an OpdB-overexpressing Escherichia coli strain, we found that the overexpressed peptidase makes the bacterial cells specifically less susceptible to several proline-rich antimicrobial peptides known to penetrate into the bacterial cytosol, and that its level of activity directly correlates with the degree of resistance. We established that E. coli OpdB can efficiently hydrolyze in vitro cationic antimicrobial peptides up to 30 residues in length, even though they contained several prolines, shortening them to inactive fragments. Two consecutive basic residues are a preferred cleavage site for the peptidase. In the case of a single basic residue, there is no cleavage if proline residues are present in the P1 and P2 positions. These results also indicate that cytosolic peptidases may cause resistance to antimicrobial peptides that have an intracellular mechanism of action, such as the proline-rich peptides, and may contribute to define the substrate specificity of the E. coli OpdB. PMID:24225368

  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. Estradiol receptor has proteolytic activity that is responsible for its own transformation.

    PubMed Central

    Puca, G A; Abbondanza, C; Nigro, V; Armetta, I; Medici, N; Molinari, A M

    1986-01-01

    We have investigated the effect of various protease inhibitors and substrates on the hormone- and temperature-dependent binding of partially purified estradiol-receptor complex to isolated nuclei. Only serine protease substrates and inhibitors significantly depressed estradiol receptor transformation. At 20 degrees C, we observed 50% inhibition with about 3 microM aprotinin or with 1.4 mM diisopropyl fluorophosphate. Aprotinin also blocked those size and charge modifications of receptor that are characteristic of the transformation process. The estradiol receptor was able to bind to aprotinin-agarose only under transforming conditions; i.e., the interaction was hormone- and temperature-dependent and inhibited by molybdate. Diisopropyl fluorophosphate, a covalent reagent for serine esterases, competitively inhibited the binding and specifically eluted the estradiol-receptor complex that had been bound to aprotinin-agarose. These results indicate that estradiol receptor transformation is due to the effect of a serine protease and that the receptor itself is endowed with this catalytic activity, which is triggered by the steroid. PMID:2426695

  13. Estradiol receptor has proteolytic activity that is responsible for its own transformation.

    PubMed

    Puca, G A; Abbondanza, C; Nigro, V; Armetta, I; Medici, N; Molinari, A M

    1986-08-01

    We have investigated the effect of various protease inhibitors and substrates on the hormone- and temperature-dependent binding of partially purified estradiol-receptor complex to isolated nuclei. Only serine protease substrates and inhibitors significantly depressed estradiol receptor transformation. At 20 degrees C, we observed 50% inhibition with about 3 microM aprotinin or with 1.4 mM diisopropyl fluorophosphate. Aprotinin also blocked those size and charge modifications of receptor that are characteristic of the transformation process. The estradiol receptor was able to bind to aprotinin-agarose only under transforming conditions; i.e., the interaction was hormone- and temperature-dependent and inhibited by molybdate. Diisopropyl fluorophosphate, a covalent reagent for serine esterases, competitively inhibited the binding and specifically eluted the estradiol-receptor complex that had been bound to aprotinin-agarose. These results indicate that estradiol receptor transformation is due to the effect of a serine protease and that the receptor itself is endowed with this catalytic activity, which is triggered by the steroid. PMID:2426695

  14. 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

  15. 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

  16. 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. PMID:24268446

  17. Proteolytic Cleavage Driven by Glycosylation.

    PubMed

    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

  18. 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. PMID:15261386

  19. C1q-TNF-related protein-9, a novel cardioprotetcive cardiokine, requires proteolytic cleavage to generate a biologically active globular domain isoform.

    PubMed

    Yuan, Yuexing; Lau, Wayne Bond; Su, Hui; Sun, Yang; Yi, Wei; Du, Yunhui; Christopher, Theodore; Lopez, Bernard; Wang, Yajing; Ma, Xin-Liang

    2015-05-15

    Prevalence and severity of postmyocardial infarction heart failure continually escalate in type 2 diabetes via incompletely understood mechanisms. The discovery of the cardiac secretomes, collectively known as "cardiokines", has significantly enhanced appreciation of the local microenvironment's influence on disease development. Recent studies demonstrated that C1q-TNF-related protein-9 (CTRP9), a newly discovered adiponectin (APN) paralog, is highly expressed in the heart. However, its relationship with APN (concerning diabetic cardiovascular injury in particular) remains unknown. Plasma CTRP9 levels are elevated in APN knockout and reduced in diabetic mice. In contrast to APN, which circulates as full-length multimers, CTRP9 circulates in the plasma primarily in the globular domain isoform (gCTRP9). Recombinant full-length CTRP9 (fCTRP9) was cleaved when incubated with cardiac tissue extracts, generating gCTRP9, a process inhibited by protease inhibitor cocktail. gCTRP9 rapidly activates cardiac survival kinases, including AMPK, Akt, and endothelial NOS. However, fCTRP9-mediated kinase activation is much less potent and significantly delayed. Kinase activation by fCTRP9, but not gCTRP9, is inhibited by protease inhibitor cocktail. These results demonstrate for the first time that the novel cardiokine CTRP9 undergoes proteolytic cleavage to generate gCTRP9, the dominant circulatory and actively cardioprotective isoform. Enhancing cardiac CTRP9 production and/or its proteolytic posttranslational modification are of therapeutic potential, attenuating diabetic cardiac injury. PMID:25783894

  20. A1M/α1-microglobulin is proteolytically activated by myeloperoxidase, binds its heme group and inhibits low density lipoprotein oxidation

    PubMed Central

    Cederlund, Martin; Deronic, Adnan; Pallon, Jan; Sørensen, Ole E.; Åkerström, Bo

    2015-01-01

    α1-microglobulin (A1M) is a 26 kDa plasma and tissue protein with reductase activity and radical- and heme-binding anti-oxidative functions. In addition, exposure of A1M to hemoglobin has been shown to induce proteolytic elimination of a C-terminal tetrapeptide yielding a heme-degrading form, truncated A1M (t-A1M). Myeloperoxidase (MPO), a heme-containing enzyme that catalyzes the production of free radicals and hypochlorite, is released by neutrophils during the inflammatory response to bacterial infections. MPO-induced low density lipoprotein (LDL)-oxidation in blood has been suggested as a causative factor in atherosclerosis. In this study we have hypothesized that A1M interacts with MPO in a similar mode as with hemoglobin, and is a regulator of its activity. The results show that A1M is proteolytically cleaved, with formation of t-A1M, after exposure to MPO, and that t-A1M contains iron and heme-degradation products. The reaction is dependent of pH, time and concentration of substrates and a pH-value around 7 is shown to be optimal for cleavage. Furthermore, A1M inhibits MPO- and hydrogen peroxide-induced oxidation of LDL. The results suggest that A1M may have a role as an inhibitor of the damaging effects of the neutrophil respiratory burst on bystander tissue components. PMID:25698971

  1. 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. PMID:26451973

  2. Regulated proteolytic processing of Reelin through interplay of tissue plasminogen activator (tPA), ADAMTS-4, ADAMTS-5, and their modulators.

    PubMed

    Krstic, Dimitrije; Rodriguez, Myriam; Knuesel, Irene

    2012-01-01

    The extracellular signaling protein Reelin, indispensable for proper neuronal migration and cortical layering during development, is also expressed in the adult brain where it modulates synaptic functions. It has been shown that proteolytic processing of Reelin decreases its signaling activity and promotes Reelin aggregation in vitro, and that proteolytic processing is affected in various neurological disorders, including Alzheimer's disease (AD). However, neither the pathophysiological significance of dysregulated Reelin cleavage, nor the involved proteases and their modulators are known. Here we identified the serine protease tissue plasminogen activator (tPA) and two matrix metalloproteinases, ADAMTS-4 and ADAMTS-5, as Reelin cleaving enzymes. Moreover, we assessed the influence of several endogenous protease inhibitors, including tissue inhibitors of metalloproteinases (TIMPs), α-2-Macroglobulin, and multiple serpins, as well as matrix metalloproteinase 9 (MMP-9) on Reelin cleavage, and described their complex interplay in the regulation of this process. Finally, we could demonstrate that in the murine hippocampus, the expression levels and localization of Reelin proteases largely overlap with that of Reelin. While this pattern remained stable during normal aging, changes in their protein levels coincided with accelerated Reelin aggregation in a mouse model of AD. PMID:23082219

  3. 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

  4. 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. PMID:23755406

  5. 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. PMID:26327595

  6. 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

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

    PubMed Central

    Tomaić, V; Banks, L

    2015-01-01

    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. PMID:25633294

  8. 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. PMID:25747830

  9. 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

    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 Arg(164). 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

  10. Effects of a potato cysteine proteinase inhibitor on midgut proteolytic enzyme activity and growth of the southern corn rootworm, Diabrotica undecimpunctata howardi (Coleoptera: Chrysomelidae).

    PubMed

    Fabrick, J; Behnke, C; Czapla, T; Bala, K; Rao, A G; Kramer, K J; Reeck, G R

    2002-04-01

    The major proteinase activity in extracts of larval midguts from the southern corn rootworm (SCR), Diabrotica undecimpunctata howardi, was identified as a cysteine proteinase that prefers substrates containing an arginine residue in the P1 position. Gelatin-zymogram analysis of the midgut proteinases indicated that the artificial diet-fed SCR, corn root-fed SCR, and root-fed western corn rootworms (Diabrotica virgifera virgifera) possess a single major proteinase with an apparent molecular mass of 25kDa and several minor proteinases. Similar proteinase activity pH profiles were exhibited by root-fed and diet-fed rootworms with the optimal activity being slightly acidic. Rootworm larvae reared on corn roots exhibited significantly less caseinolytic activity than those reared on the artificial diet. Midgut proteolytic activity from SCR was most sensitive to inhibition by inhibitors of cysteine proteinases. Furthermore, rootworm proteinase activity was particularly sensitive to inhibition by a commercial protein preparation from potato tubers (PIN-II). One of the proteins, potato cysteine proteinase inhibitor-10', PCPI-10', obtained from PIN-II by ion-exchange chromatography, was the major source of inhibitory activity against rootworm proteinase activity. PCPI-10' and E-64 were of comparable potency as inhibitors of southern corn rootworm proteinase activity (IC(50) =31 and 35nM, respectively) and substantially more effective than chicken egg white cystatin (IC(50) =121nM). Incorporation of PCPI-10' into the diet of SCR larvae in feeding trials resulted in a significant increase in mortality and growth inhibition. We suggest that expression of inhibitors such as PCPI-10' by transgenic corn plants in the field is a potentially attractive method of host plant resistance to these Diabrotica species. PMID:11886775

  11. 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.

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

    PubMed Central

    2011-01-01

    Background 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. Methods 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. Results 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). Conclusion 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. PMID:21211028

  13. Active Site Detection by Spatial Conformity and Electrostatic Analysis—Unravelling a Proteolytic Function in Shrimp Alkaline Phosphatase

    PubMed Central

    Chakraborty, Sandeep; Minda, Renu; Salaye, Lipika; Bhattacharjee, Swapan K.; Rao, Basuthkar J.

    2011-01-01

    Computational methods are increasingly gaining importance as an aid in identifying active sites. Mostly these methods tend to have structural information that supplement sequence conservation based analyses. Development of tools that compute electrostatic potentials has further improved our ability to better characterize the active site residues in proteins. We have described a computational methodology for detecting active sites based on structural and electrostatic conformity - CataLytic Active Site Prediction (CLASP). In our pipelined model, physical 3D signature of any particular enzymatic function as defined by its active sites is used to obtain spatially congruent matches. While previous work has revealed that catalytic residues have large pKa deviations from standard values, we show that for a given enzymatic activity, electrostatic potential difference (PD) between analogous residue pairs in an active site taken from different proteins of the same family are similar. False positives in spatially congruent matches are further pruned by PD analysis where cognate pairs with large deviations are rejected. We first present the results of active site prediction by CLASP for two enzymatic activities - β-lactamases and serine proteases, two of the most extensively investigated enzymes. The results of CLASP analysis on motifs extracted from Catalytic Site Atlas (CSA) are also presented in order to demonstrate its ability to accurately classify any protein, putative or otherwise, with known structure. The source code and database is made available at www.sanchak.com/clasp/. Subsequently, we probed alkaline phosphatases (AP), one of the well known promiscuous enzymes, for additional activities. Such a search has led us to predict a hitherto unknown function of shrimp alkaline phosphatase (SAP), where the protein acts as a protease. Finally, we present experimental evidence of the prediction by CLASP by showing that SAP indeed has protease activity in vitro. PMID

  14. Neutrophilic Cathepsin C Is Maturated by a Multistep Proteolytic Process and Secreted by Activated Cells during Inflammatory Lung Diseases.

    PubMed

    Hamon, Yveline; Legowska, Monika; Hervé, Virginie; Dallet-Choisy, Sandrine; Marchand-Adam, Sylvain; Vanderlynden, Lise; Demonte, Michèle; Williams, Rich; Scott, Christopher J; Si-Tahar, Mustapha; Heuzé-Vourc'h, Nathalie; Lalmanach, Gilles; Jenne, Dieter E; Lesner, Adam; Gauthier, Francis; Korkmaz, Brice

    2016-04-15

    The cysteine protease cathepsin C (CatC) activates granule-associated proinflammatory serine proteases in hematopoietic precursor cells. Its early inhibition in the bone marrow is regarded as a new therapeutic strategy for treating proteolysis-driven chronic inflammatory diseases, but its complete inhibition is elusive in vivo Controlling the activity of CatC may be achieved by directly inhibiting its activity with a specific inhibitor or/and by preventing its maturation. We have investigated immunochemically and kinetically the occurrence of CatC and its proform in human hematopoietic precursor cells and in differentiated mature immune cells in lung secretions. The maturation of proCatC obeys a multistep mechanism that can be entirely managed by CatS in neutrophilic precursor cells. CatS inhibition by a cell-permeable inhibitor abrogated the release of the heavy and light chains from proCatC and blocked ∼80% of CatC activity. Under these conditions the activity of neutrophil serine proteases, however, was not abolished in precursor cell cultures. In patients with neutrophilic lung inflammation, mature CatC is found in large amounts in sputa. It is secreted by activated neutrophils as confirmed through lipopolysaccharide administration in a nonhuman primate model. CatS inhibitors currently in clinical trials are expected to decrease the activity of neutrophilic CatC without affecting those of elastase-like serine proteases. PMID:26884336

  15. 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…

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  17. 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. PMID:22610687

  18. Effect of legume seed extracts on the inhibition of proteolytic activity and muscle degradation of fresh water prawn (Macrobrachiumrosenbergii).

    PubMed

    Sriket, Chodsana; Benjakul, Soottawat; Visessanguan, Wonnop; Hara, Kenji

    2011-12-01

    Trypsin inhibitors in the extracts from soybean (Glycine max), adzuki bean (Vigna angularis), bambara groundnut (Vigna subterranea) and red kidney bean (Phaseoulus vulgaris) varied in amount and molecular weight. The soybean extract had the highest level of trypsin inhibitor with molecular weight (MW) of 21kDa, followed by bambara groundnut extract possessing trypsin inhibitor with MW of 15kDa. Both extracts showed a more effective inhibition towards crude protease extract (CE) from the hepatopancreas of fresh water prawn (Macrobrachium rosenbergii) than the extracts from adzuki and red kidney beans. Activity staining also reconfirmed the higher inhibitory activity on CE from hepatopancreas by the extracts from both soybean and bambara groundnut. The extracts from all seeds were able to inhibit the degradation of fresh water prawn meat containing CE in a concentration dependent manner. Based on inhibitor study, the extracts from soybean and bambara groundnut can be a potential aid to suppress the muscle softening of fresh water prawn, mediated by trypsin-like proteases released from hepatopancreas, during extended iced storage. PMID:25212342

  19. The Unusual Resistance of Avian Defensin AvBD7 to Proteolytic Enzymes Preserves Its Antibacterial Activity.

    PubMed

    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

  20. 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

  1. 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. PMID:26876839

  2. 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. PMID:25447774

  3. 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

  4. 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

  5. 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

  6. 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

  7. The 53-kDa proteolytic product of precursor starch-hydrolyzing enzyme of Aspergillus niger has Taka-amylase-like activity.

    PubMed

    Ravi-Kumar, K; Venkatesh, K S; Umesh-Kumar, S

    2007-04-01

    The 53-kDa amylase secreted by Aspergillus niger due to proteolytic processing of the precursor starch-hydrolyzing enzyme was resistant to acarbose, a potent alpha-glucosidase inhibitor. The enzyme production was induced when A. niger was grown in starch medium containing the inhibitor. Antibodies against the precursor enzyme cross-reacted with the 54-kDa Taka-amylase protein of A. oryzae. It resembled Taka-amylase in most of its properties and also hydrolyzed starch to maltose of alpha-anomeric configuration. However, it did not degrade maltotriose formed during the reaction and was not inhibited by zinc ions. PMID:17123073

  8. Tumor Necrosis Factor-α-induced Proteolytic Activation of Pro-matrix Metalloproteinase-9 by Human Skin Is Controlled by Down-regulating Tissue Inhibitor of Metalloproteinase-1 and Mediated by Tissue-associated Chymotrypsin-like Proteinase*

    PubMed Central

    Han, Yuan-Ping; Nien, Yih-Dar; Garner, Warren L.

    2008-01-01

    The proteolytic activation of pro-matrix metalloproteinase (MMP)-9 by conversion of the 92-kDa precursor into an 82-kDa active form has been observed in chronic wounds, tumor metastasis, and many inflammation-associated diseases, yet the mechanistic pathway to control this process has not been identified. In this report, we show that the massive expression and activation of MMP-9 in skin tissue from patients with chronically unhealed wounds could be reconstituted in vitro with cultured normal human skin by stimulation with transforming growth factor-β and tumor necrosis factor (TNF)-α. We dissected the mechanistic pathway for TNF-α induced activation of pro-MMP-9 in human skin. We found that proteolytic activation of pro-MMP-9 was mediated by a tissue-associated chymotrypsin-like proteinase, designated here as pro-MMP-9 activator (pM9A). This unidentified activator specifically converted pro-MMP-9 but not pro-MMP-2, another member of the gelatinase family. The tissue-bound pM9A was steadily expressed and not regulated by TNF-α, which indicated that the cytokine-mediated activation of pro-MMP-9 might be regulated at the inhibitor level. Indeed, the skin constantly secreted tissue inhibitor of metalloproteinase-1 at the basal state. TNF-α, but not transforming growth factor-β, down-regulated this inhibitor. The TNF-α-mediated activation of pro-MMP-9 was tightly associated with down-regulation of tissue inhibitor of metalloproteinase-1 in a dose-dependent manner. To establish this linkage, we demonstrate that the recombinant tissue inhibitor of metalloproteinase-1 could block the activation of pro-MMP-9 by either the intact skin or skin fractions. Thus, these studies suggest a novel regulation for the proteolytic activation of MMP-9 in human tissue, which is mediated by tissue-bound activator and controlled by down-regulation of a specific inhibitor. PMID:12004062

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. 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. PMID:26822063

  14. Activity-Modulating Monoclonal Antibodies to the Human Serine Protease HtrA3 Provide Novel Insights into Regulating HtrA Proteolytic Activities

    PubMed Central

    Singh, Harmeet; Nero, Tracy L.; Wang, Yao; Parker, Michael W.; Nie, Guiying

    2014-01-01

    Mammalian HtrA (high temperature requirement factor A) proteases, comprising 4 multi-domain members HtrA1-4, play important roles in a number of normal cellular processes as well as pathological conditions such as cancer, arthritis, neurodegenerative diseases and pregnancy disorders. However, how HtrA activities are regulated is not well understood, and to date no inhibitors specific to individual HtrA proteins have been identified. Here we investigated five HtrA3 monoclonal antibodies (mAbs) that we have previously produced, and demonstrated that two of them regulated HtrA3 activity in an opposing fashion: one inhibited while the other stimulated. The inhibitory mAb also blocked HtrA3 activity in trophoblast cells and enhanced migration and invasion, confirming its potential in vivo utility. To understand how the binding of these mAbs modulated HtrA3 protease activity, their epitopes were visualized in relation to a 3-dimensional HtrA3 homology model. This model suggests that the inhibitory HtrA3 mAb blocks substrate access to the protease catalytic site, whereas the stimulatory mAb may bind to the PDZ domain alone or in combination with the N-terminal and protease domains. Since HtrA1, HtrA3 and HtrA4 share identical domain organization, our results establish important foundations for developing potential therapeutics to target these HtrA proteins specifically for the treatment of a number of diseases, including cancer and pregnancy disorders. PMID:25248123

  15. 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.

  16. 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.

  17. 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

  18. Proteolytic components of serum IgG preparations

    PubMed Central

    Li, L; Kalaga, R; Paul, S

    2000-01-01

    Chemical catalysis, an effector mechanism utilized by fully assembled antibodies, can also be mediated by the isolated antibody subunits. Because trace amounts of free light chains (L chains) are present in IgG preparations, a detailed study was undertaken to identify the constituents responsible for the polyreactive proteolytic activity of IgG purified from human sera, determined as the extent of cleavage of the model peptide substrate Pro-Phe-Arg-methylcoumarinamide. Two proteolytic species with approximate mass of 50 kD and 150 kD were separated by repetitive gel filtration in a denaturing solvent (6 m guanidine hydrochloride). The activity of the renatured 50-kD fraction (in fluorescence units/μg protein) was more than 45-fold greater than of the 150-kD fraction. Both fractions lost the activity following immunoadsorption on immobilized anti-IgG antibody. Fab fragments prepared from the 150-kD IgG fraction retained the activity. Reducing and non-reducing SDS-electrophoresis suggested the 50-kD fraction isolated from the IgG preparations to be a mixture of heavy chain (H chain) monomers and disulphide bonded L chain dimers. Electrophoretically homogeneous monomers of 50-kD H chains and 25-kD L chains were prepared by gel filtration of reduced and alkylated IgG from seven human subjects. Each of the alkylated L chain preparations displayed the proteolytic activity. The activity in alkylated H chains was undetectable or only marginally greater than the background values. L chain dimers appear to be the major species responsible for the polyreactive proteolytic activity of serum IgG preparations, with a smaller contribution furnished by tetrameric IgG. PMID:10792374

  19. 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. PMID:18800269

  20. Activation of an Mg2+-dependent DNA endonuclease of avian myeloblastosis virus alpha beta DNA polymerase by in vitro proteolytic cleavage.

    PubMed Central

    Grandgenett, D P; Golomb, M; Vora, A C

    1980-01-01

    Partial chymotryptic digestion of purified avian myeloblastosis virus alpha beta DNA polymerase resulted in the activation of a Mg2+-dependent DNA endonuclease activity. Incubation of the polymerase-protease mixture in the presence of super-coiled DNA and Mg2+ permitted detection of the cleaved polymerase fragment possessing DNA nicking activity. Protease digestion conditions were established permitting selective cleavage of beta to alpha, which contained DNA polymerase and RNase H activity and to a family of polypeptides ranging in size from 30,000 to 34,000 daltons. These latter beta-unique fragments were purified by polyuridylate-Sepharose 4B chromatography and were shown to contain both DNA binding and DNA endonuclease activities. We have demonstrated that this group of polymerase fragments derived by chymotryptic digestion of alpha beta DNA polymerase is similar to the in vivo-isolated avian myeloblastosis virus p32pol in size, sequence, and DNA endonuclease activity. Images PMID:6154149

  1. Radiation inactivation analysis of assimilatory NADH:nitrate reductase. Apparent functional sizes of partial activities associated with intact and proteolytically modified enzyme

    SciTech Connect

    Solomonson, L.P.; McCreery, M.J.; Kay, C.J.; Barber, M.J.

    1987-06-25

    Recently we demonstrated that target sizes for the partial activities of nitrate reductase were considerably smaller than the 100-kDa subunit which corresponded to the target size of the full (physiologic) activity NADH:nitrate reductase. These results suggested that the partial activities resided on functionally independent domains and that radiation inactivation may be due to localized rather than extensive damage to protein structure. The present study extends these observations and addresses several associated questions. Monophasic plots were observed over a wide range of radiation doses, suggesting a single activity component in each case. No apparent differences were observed over a 10-fold range of concentration for each substrate, suggesting that the observed slopes were not due to marked changes in Km values. Apparent target sizes estimated for partial activities associated with native enzyme and with limited proteolysis products of native enzyme suggested that the functional size obtained by radiation inactivation analysis is independent of the size of the polypeptide chain. The presence of free radical scavengers during irradiation reduced the apparent target size of both the physiologic and partial activities by an amount ranging from 24 to 43%, suggesting that a free radical mechanism is at least partially responsible for the inactivation. Immunoblot analysis of nitrate reductase irradiated in the presence of free radical scavengers revealed formation of distinct bands at 90, 75, and 40 kDa with increasing doses of irradiation rather than complete destruction of the polypeptide chain.

  2. [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). PMID:26065261

  3. 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. PMID:25176510

  4. 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

  5. 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

  6. Quantum-dot-based nanosensors designed for proteolytic monitoring

    NASA Astrophysics Data System (ADS)

    Medintz, Igor L.; Clapp, Aaron R.; Brunel, Florence M.; Goldman, Ellen R.; Chang, Eddie L.; Dawson, Phillip E.; Mattoussi, Hedi

    2006-02-01

    We have previously assembled QD-based fluorescence resonance energy transfer (FRET) sensors specific for the sugar nutrient maltose and the explosive TNT. These sensors utilize several inherent benefits of QDs as FRET donors. In this report, we show that QD-FRET based sensors can also function in the monitoring of proteolytic enzyme activity. We utilize a QD with multiple dye-labeled proteins attached to the surface as a substrate for a prototypical protease. We then demonstrate how this strategy can be extended to detect protease activity by utilizing a dye-labeled peptide attached to the QD as a proteolytic substrate. Self-assembly of the peptide-dye on the QD brings the dye in close proximity to the QD and result in efficient FRET. Addition of a proteolytic enzyme that specifically recognizes and cleaves the peptide alters the FRET signature of the sensor in a concentration-dependent manner. Both qualitative and quantitative data can be derived from these sensors. The potential benefits of this type of QD sensing strategy are discussed.

  7. New Ligand Binding Function of Human Cerberus and Role of Proteolytic Processing in Regulating Ligand-Receptor Interactions and Antagonist Activity.

    PubMed

    Aykul, Senem; Martinez-Hackert, Erik

    2016-02-13

    Cerberus is a key regulator of vertebrate embryogenesis. Its biological function has been studied extensively in frog and mouse embryos. Its ability to bind and antagonize the transforming growth factor-β (TGF-β) family ligand Nodal is well established. Strikingly, the molecular function of Cerberus remains poorly understood. The underlying reason is that Cerberus is a complex, multifunctional protein: It binds and inhibits multiple TGF-β family ligands, it may bind and inhibit some Wnt family members, and two different forms with distinct activities have been described. In addition, sequence homology between frog and mammalian Cerberus is low, suggesting that previous studies, which analyzed frog Cerberus function, may not accurately describe the function of mammalian Cerberus. We therefore undertook to determine the molecular activities of human Cerberus in TGF-β family signaling. Using purified proteins, surface plasmon resonance, and reporter gene assays, we discovered that human Cerberus bound and inhibited the TGF-β family ligands Activin B, BMP-6, and BMP-7, but not the frog Cerberus ligand BMP-2. Notably, full-length Cerberus successfully blocked ligand binding to type II receptors, but the short form was less effective. In addition, full-length Cerberus suppressed breast cancer cell migration but the short form did not. Thus, our findings expand the roles of Cerberus as TGF-β family signaling inhibitor, provide a molecular rationale for the function of the N-terminal region, and support the idea that Cerberus could have regulatory activities beyond direct inhibition of TGF-β family signaling. PMID:26802359

  8. Protease Activity of PprI Facilitates DNA Damage Response: Mn(2+)-Dependence and Substrate Sequence-Specificity of the Proteolytic Reaction

    PubMed Central

    Lu, Huiming; Lin, Lin; Wang, Liangyan; Xu, Hong; Cui, Xianyan; Zhang, Hui; Li, Tingting; Hua, Yuejin

    2015-01-01

    The extremophilic bacterium Deinococcus radiodurans exhibits an extraordinary resistance to ionizing radiation. Previous studies established that a protein named PprI, which exists only in the Deinococcus-Thermus family, acts as a general switch to orchestrate the expression of a number of DNA damage response (DDR) proteins involved in cellular radio-resistance. Here we show that the regulatory mechanism of PprI depends on its Mn(2+)-dependent protease activity toward DdrO, a transcription factor that suppresses DDR genes’ expression. Recognition sequence-specificity around the PprI cleavage site is essential for DNA damage repair in vivo. PprI and DdrO mediate a novel DNA damage response pathway differing from the classic LexA-mediated SOS response system found in radiation-sensitive bacterium Escherichia coli. This PprI-mediated pathway in D. radiodurans is indispensable for its extreme radio-resistance and therefore its elucidation significantly advances our understanding of the DNA damage repair mechanism in this amazing organism. PMID:25811789

  9. Protease activity of PprI facilitates DNA damage response: Mn2+-dependence and substrate sequence-specificity of the proteolytic reaction.

    PubMed

    Wang, Yunguang; Xu, Qiang; Lu, Huiming; Lin, Lin; Wang, Liangyan; Xu, Hong; Cui, Xianyan; Zhang, Hui; Li, Tingting; Hua, Yuejin

    2015-01-01

    The extremophilic bacterium Deinococcus radiodurans exhibits an extraordinary resistance to ionizing radiation. Previous studies established that a protein named PprI, which exists only in the Deinococcus-Thermus family, acts as a general switch to orchestrate the expression of a number of DNA damage response (DDR) proteins involved in cellular radio-resistance. Here we show that the regulatory mechanism of PprI depends on its Mn(2+)-dependent protease activity toward DdrO, a transcription factor that suppresses DDR genes' expression. Recognition sequence-specificity around the PprI cleavage site is essential for DNA damage repair in vivo. PprI and DdrO mediate a novel DNA damage response pathway differing from the classic LexA-mediated SOS response system found in radiation-sensitive bacterium Escherichia coli. This PprI-mediated pathway in D. radiodurans is indispensable for its extreme radio-resistance and therefore its elucidation significantly advances our understanding of the DNA damage repair mechanism in this amazing organism. PMID:25811789

  10. 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. PMID:25623068

  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. PMID:27460704

  12. 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. PMID:26989505

  13. 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

  14. 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.

  15. Proteolytic crosstalk in multi-protease networks.

    PubMed

    Ogle, Curtis T; Mather, William H

    2016-01-01

    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. PMID:27042892

  16. Effect of dietary fiber on proteolytic pancreatic enzymes in vitro.

    PubMed

    Hansen, W E

    1986-12-01

    Chymotrypsin, trypsin, carboxypeptidase A and B, elastase and enterokinase activities were measured in buffer solutions and in human duodenal juice after incubation with wheat bran, cellulose, guar gum, pectin, psyllium and lignin. The different types of dietary fiber led to inhibition of enzymatic activity in most experiments, e.g., lignin could totally ablish the activity of isolated trypsin and chymotrypsin. Only in enterokinase was there no influence. Inhibition depended on incubation time; the effect was proportional to fiber concentration and inversely related to enzyme level. Treatment of fiber with hydrochloric acid (pH 1.5) and heat (95 degrees C) destroyed inhibitory activity in some experiments. The effect of lignin on one enzyme (trypsin) was reduced by the addition of another enzyme (chymotrypsin). It is concluded that dietary fiber could affect digestion by inhibiting proteolytic pancreatic enzymes. PMID:2824629

  17. Proteolytic regulation of stress response pathways in Escherichia coli.

    PubMed

    Micevski, Dimce; Dougan, David A

    2013-01-01

    Maintaining correct cellular function is a fundamental biological process for all forms of life. A critical aspect of this process is the maintenance of protein homeostasis (proteostasis) in the cell, which is largely performed by a group of proteins, referred to as the protein quality control (PQC) network. This network of proteins, comprised of chaperones and proteases, is critical for maintaining proteostasis not only during favourable growth conditions, but also in response to stress. Indeed proteases play a crucial role in the clearance of unwanted proteins that accumulate during stress, but more importantly, in the activation of various different stress response pathways. In bacteria, the cells response to stress is usually orchestrated by a specific transcription factor (sigma factor). In Escherichia coli there are seven different sigma factors, each of which responds to a particular stress, resulting in the rapid expression of a specific set of genes. The cellular concentration of each transcription factor is tightly controlled, at the level of transcription, translation and protein stability. Here we will focus on the proteolytic regulation of two sigma factors (σ(32) and σ(S)), which control the heat and general stress response pathways, respectively. This review will also briefly discuss the role proteolytic systems play in the clearance of unwanted proteins that accumulate during stress. PMID:23479439

  18. 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

  19. 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. PMID:26798196

  20. Mate (Ilex paraguariensis St. Hilaire) saponins induce caspase-3-dependent apoptosis in human colon cancer cells in vitro

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  1. 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. PMID:26931430

  2. Proteolytic Enzymes Clustered in Specialized Plasma-Membrane Domains Drive Endothelial Cells’ Migration

    PubMed Central

    Salamone, Monica; Carfì Pavia, Francesco

    2016-01-01

    In vitro cultured endothelial cells forming a continuous monolayer establish stable cell-cell contacts and acquire a “resting” phenotype; on the other hand, when growing in sparse conditions these cells acquire a migratory phenotype and invade the empty area of the culture. Culturing cells in different conditions, we compared expression and clustering of proteolytic enzymes in cells having migratory versus stationary behavior. In order to observe resting and migrating cells in the same microscopic field, a continuous cell monolayer was wounded. Increased expression of proteolytic enzymes was evident in cell membranes of migrating cells especially at sprouting sites and in shed membrane vesicles. Gelatin zymography and western blotting analyses confirmed that in migrating cells, expression of membrane-bound and of vesicle-associated proteolytic enzymes are increased. The enzymes concerned include MMP-2, MMP-9, MT1-MMP, seprase, DPP4 (DiPeptidyl Peptidase 4) and uPA. Shed membrane vesicles were shown to exert degradative activity on ECM components and produce substrates facilitating cell migration. Vesicles shed by migrating cells degraded ECM components at an increased rate; as a result their effect on cell migration was amplified. Inhibiting either Matrix Metallo Proteases (MMPs) or Serine Integral Membrane Peptidases (SIMPs) caused a decrease in the stimulatory effect of vesicles, inhibiting the spontaneous migratory activity of cells; a similar result was also obtained when a monoclonal antibody acting on DPP4 was tested. We conclude that proteolytic enzymes have a synergistic stimulatory effect on cell migration and that their clustering probably facilitates the proteolytic activation cascades needed to produce maximal degradative activity on cell substrates during the angiogenic process. PMID:27152413

  3. TAILS N-terminomics of human platelets reveals pervasive metalloproteinase-dependent proteolytic processing in storage.

    PubMed

    Prudova, Anna; Serrano, Katherine; Eckhard, Ulrich; Fortelny, Nikolaus; Devine, Dana V; Overall, Christopher M

    2014-12-18

    Proteases, and specifically metalloproteinases, have been linked to the loss of platelet function during storage before transfusion, but the underlying mechanisms remain unknown. We used a dedicated N-terminomics technique, iTRAQ terminal amine isotopic labeling of substrates (TAILS), to characterize the human platelet N-terminome, proteome, and posttranslational modifications throughout platelet storage over 9 days under blood-banking conditions. From the identified 2938 proteins and 7503 unique peptides, we characterized N-terminal methionine excision, co- and posttranslational Nα acetylation, protein maturation, and proteolytic processing of proteins in human platelets. We also identified for the first time 10 proteins previously classified by the Human Proteome Organization as "missing" in the human proteome. Most N termini (77%) were internal neo-N termini (105 were novel potential alternative translation start sites, and 2180 represented stable proteolytic products), thus highlighting a prominent yet previously uncharacterized role of proteolytic processing during platelet storage. Protease inhibitor studies revealed metalloproteinases as being primarily responsible for proteolytic processing (as opposed to degradation) during storage. System-wide identification of metalloproteinase and other proteinase substrates and their respective cleavage sites suggests novel mechanisms of the effect of proteases on protein activity and platelet function during storage. All data sets and metadata are available through ProteomeXchange with the data set identifier PXD000906. PMID:25331112

  4. The calcium-dependent proteolytic system calpain-calpastatin in Drosophila melanogaster.

    PubMed Central

    Pintér, M; Friedrich, P

    1988-01-01

    Ca2+-dependent proteolytic activity was detected at pH 7.5 in head extracts of the fruit fly Drosophila melanogaster. This activity was abolished by iodoacetate, but was unaffected by phenylmethanesulphonyl fluoride. These properties resemble those of the Ca2+-dependent thiol-proteinase calpain. The activity appeared at Mr 280,000 on Sepharose CL-6B gel chromatography. DEAE-cellulose chromatography revealed two activity peaks, with elution positions corresponding to vertebrate calpains I and II. The fly head enzymes were inhibited by a heat-stable and trypsin-sensitive component of the fly head extract, which also inhibited calpains from rat kidney. The inhibitor emerged from Sepharose CL-6B columns at Mr 310,000 and from DEAE-cellulose at a position corresponding to the protein inhibitor calpastatin from other sources. It is concluded that Drosophila heads comprise the Ca2+-dependent calpain-calpastatin proteolytic system. PMID:2845920

  5. Sequence-derived structural features driving proteolytic processing.

    PubMed

    Belushkin, Alexander A; Vinogradov, Dmitry V; Gelfand, Mikhail S; Osterman, Andrei L; Cieplak, Piotr; Kazanov, Marat D

    2014-01-01

    Proteolytic signaling, or regulated proteolysis, is an essential part of many important pathways such as Notch, Wnt, and Hedgehog. How the structure of the cleaved substrate regions influences the efficacy of proteolytic processing remains underexplored. Here, we analyzed the relative importance in proteolysis of various structural features derived from substrate sequences using a dataset of more than 5000 experimentally verified proteolytic events captured in CutDB. Accessibility to the solvent was recognized as an essential property of a proteolytically processed polypeptide chain. Proteolytic events were found nearly uniformly distributed among three types of secondary structure, although with some enrichment in loops. Cleavages in α-helices were found to be relatively abundant in regions apparently prone to unfolding, while cleavages in β-structures tended to be located at the periphery of β-sheets. Application of the same statistical procedures to proteolytic events divided into separate sets according to the catalytic classes of proteases proved consistency of the results and confirmed that the structural mechanisms of proteolysis are universal. The estimated prediction power of sequence-derived structural features, which turned out to be sufficiently high, presents a rationale for their use in bioinformatic prediction of proteolytic events. PMID:24227478

  6. SEQUENCE-DERIVED STRUCTURAL FEATURES DRIVING PROTEOLYTIC PROCESSING

    PubMed Central

    Belushkin, Alexander A.; Vinogradov, Dmitry V.; Gelfand, Mikhail S.; Osterman, Andrei L.; Cieplak, Piotr; Kazanov, Marat D.

    2014-01-01

    Proteolytic signaling, or regulated proteolysis, is an essential part of many important pathways such as Notch, Wnt, and Hedgehog. How the structure of the cleaved substrate regions influences the efficacy of proteolytic processing remains underexplored. Here, we analyzed the relative importance in proteolysis of various structural features derived from substrate sequences using a dataset of more than five thousand experimentally verified proteolytic events captured in CutDB. Accessibility to the solvent was recognized as an essential property of a proteolytically processed polypeptide chain. Proteolytic events were found nearly uniformly distributed among three types of secondary structure, although with some enrichment in loops. Cleavages in α-helices were found to be relatively abundant in regions apparently prone to unfolding, while cleavages in β-structures tended to be located at the periphery of β-sheets. Application of the same statistical procedures to proteolytic events divided into separate sets according to the catalytic classes of proteases proved consistency of the results and confirmed that the structural mechanisms of proteolysis are universal. The estimated prediction power of sequence-derived structural features, which turned out to be sufficiently high, presents a rationale for their use in bioinformatic prediction of proteolytic events. PMID:24227478

  7. 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

  8. 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

  9. 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

  10. Intestinal Absorption of Fibrinolytic and Proteolytic Lumbrokinase Extracted from Earthworm, Eisenia andrei.

    PubMed

    Yan, Xiang Mei; Kim, Chung-Hyo; Lee, Chul Kyu; Shin, Jang Sik; Cho, Il Hwan; Sohn, Uy Dong

    2010-04-01

    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

  11. Curcumin cross-linked collagen aerogels with controlled anti-proteolytic and pro-angiogenic efficacy.

    PubMed

    Dharunya, G; Duraipandy, N; Lakra, Rachita; Korapatti, Purna Sai; Jayavel, R; Kiran, Manikantan Syamala

    2016-01-01

    This paper elucidates the development of a curcumin cross-linked collagen aerogel system with controlled anti-proteolytic activity and pro-angiogenic efficacy. The results of this study showed that in situ cross-linking of curcumin with collagen leads to the development of aerogels with enhanced physical and mechanical properties. The integrity of collagen after cross-linking with curcumin was studied via FTIR spectroscopy. The results confirmed that the cross-linking with curcumin did not induce any structural changes in the collagen. The curcumin cross-linked collagen aerogels exhibited potent anti-proteolytic and anti-microbial activity. Scanning electron and atomic force microscopic analysis of curcumin cross-linked collagen aerogels showed a 3D microstructure that enhanced the adhesion and proliferation of cells. The highly organized geometry of collagen-curcumin aerogels enhanced the permeability and water-retaining ability required for the diffusion of nutrients that aid cellular growth. The pro-angiogenic properties of collagen-curcumin aerogels were ascribed to the cumulative effect of the nutraceutical and the collagen molecule, which augmented the restoration of damaged tissue. Further, these aerogels exhibited controlled anti-proteolytic activity, which makes them suitable 3D scaffolds for biomedical applications. This study provides scope for the development of biocompatible and bioresorbable collagen aerogel systems that use a nutraceutical as a cross-linker for biomedical applications. PMID:27509047

  12. 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. PMID:26436560

  13. Interaction of Leptospira interrogans with Human Proteolytic Systems Enhances Dissemination through Endothelial Cells and Protease Levels

    PubMed Central

    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.

    2013-01-01

    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. PMID:23478319

  14. 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. PMID:15700759

  15. The Caspase Proteolytic System in Callipyge and Normal Lambs in Longissimus, Semimembranosus, and Infraspinatus Muscles During Postmortem Storage

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  16. The genetic and molecular bases of monogenic disorders affecting proteolytic systems

    PubMed Central

    Richard, I

    2005-01-01

    Complete and limited proteolysis represents key events that regulate many biological processes. At least 5% of the human genome codes for components of proteolytic processes if proteases, inhibitors, and cofactors are taken into account. Accordingly, disruption of proteolysis is involved in numerous pathological conditions. In particular, molecular genetic studies have identified a growing number of monogenic disorders caused by mutations in protease coding genes, highlighting the importance of this class of enzymes in development, organogenesis, immunity, and brain function. This review provides insights into the current knowledge about the molecular genetic causes of these disorders. It should be noted that most are due to loss of function mutations, indicating absolute requirement of proteolytic activities for normal cellular functions. Recent progress in understanding the function of the implicated proteins and the disease pathogenesis is detailed. In addition to providing important clues to the diagnosis, treatment, and pathophysiology of disease, functional characterisation of mutations in proteolytic systems emphasises the pleiotropic functions of proteases in the body homeostasis. PMID:15994873

  17. Composition and proteolytic processing of corneal deposits associated with mutations in the TGFBI gene

    PubMed Central

    Karring, Henrik; Runager, Kasper; Thøgersen, Ida B.; Klintworth, Gordon K.; Højrup, Peter; Enghild, Jan J.

    2012-01-01

    Different types of granular corneal dystrophy (GCD)1 and lattice corneal dystrophy (LCD) are associated with mutations in the transforming growth factor beta induced gene (TGFBI). These dystrophies are characterized by the formation of non-amyloid granular deposits (GCDs) and amyloid (LCD type 1 and its variants) in the cornea. Typical corneal non-amyloid deposits from GCD type 2 (R124H), amyloid from a variant of LCD type 1 (V624M) and disease-free tissue controls were procured by laser capture microdissection and analyzed by tandem mass spectrometry. Label-free quantitative comparisons of deposits and controls suggested that the non-amyloid sample (R124H) specifically accumulated transforming growth factor beta induced protein (TGFBIp/keratoepithelin/βig-h3), serum amyloid P-component, clusterin, type III collagen, keratin 3, and histone H3-like protein. The amyloid (V624M) similarly accumulated serum amyloid P-component and clusterin but also a C-terminal fragment of TGFBIp containing residues Y571-R588 derived from the fourth fasciclin-1 domain (FAS1-4), apolipoprotein E and apolipoprotein A-IV. Significantly, analyses of the amyloid sample also revealed the presence of the serine protease Htr (High-temperature requirement) A1 and a number of proteolytic cleavage sites in the FAS1-4 domain of TGFBIp. These cleavage sites were consistent with the ligand binding and proteolytic activity of HtrA1 suggesting that it plays a role in the proteolytic processing of the amyloidogenic FAS1-4 domain. Taken together, the data suggest that the amyloidogenic-prone region of the fourth FAS1 domain of TGFBIp encompasses the Y571-R588 peptide and that HtrA1 is involved in the proteolytic processing of TGFBIp-derived amyloid in vivo. PMID:22155582

  18. Proteolytic cleavage and shedding of the bovine prion protein in two cell culture systems.

    PubMed

    Zhao, Hongxing; Klingeborn, Mikael; Simonsson, Magnus; Linné, Tommy

    2006-01-01

    We have compared the processing, turnover and release of bovine PrP (boPrP) in transfected baby hamster kidney (BHK) and mouse neuroblastoma (N2a) cells. In BHK cells, boPrP was subjected to two distinct proteolytic cleavage events, the first was mapped between K(121) and H(122) generating an N-terminal and a C-terminal PrP fragment. Transport block experiments, cell surface biotinylation and PIPLC analyses showed that the bulk of boPrP on the cell surface was the C-terminal fragment and indicated that the first cleavage of boPrP took place prior to or very soon after it appears at the cell surface. The second cleavage was situated at the extreme C-terminus of the boPrP GPI-anchored C-terminal fragment and as a result of this was shed into the medium rapidly. The kinetics, the migration in SDS-PAGE of the released fragment and protease inhibition studies indicate that a proteolytic activity was responsible for the release of the boPrP fragment from its GPI-anchor. Both N- and C-terminal fragments of boPrP could be detected in the medium. Moreover, in normal bovine brain, a C-terminal fragment was identified, suggesting that similar proteolytic processing events occur in vivo. In N2a cells, the majority of boPrP was subjected to a more complete degradation process, and only trace amounts of full length boPrP was shed into cell culture medium in a process which also indicated a release by proteolytic cleavage. PMID:16140411

  19. Proteolytic Processing Causes Extensive Heterogeneity of Tissue Matrilin Forms*

    PubMed Central

    Ehlen, Harald W. A.; Sengle, Gerhard; Klatt, Andreas R.; Talke, Anja; Müller, Stefan; Paulsson, Mats; Wagener, Raimund

    2009-01-01

    The matrilins are a family of multidomain extracellular matrix proteins with adapter functions. The oligomeric proteins have a bouquet-like structure and bind to a variety of different ligands whereby the avidity of their interactions is dependent on the number of subunits and domains present. Here we show the contribution of post-translational proteolytic processing to the heterogeneity of matrilins seen in tissue extracts and cell culture supernatants. A cleavage site after two glutamate residues in the hinge region close to the C-terminal coiled-coil oligomerization domain is conserved among the matrilins. Cleavage at this site yields molecules that lack almost complete subunits. The processing is least pronounced in matrilin-1 and particularly complex in matrilin-2, which contains additional cleavage sites. Replacement of the hinge region in matrilin-4 by the matrilin-1 hinge region had no marked effect on the processing. A detailed study revealed that matrilin-4 is processed already in the secretory pathway and that the activation of the responsible enzymes is dependent on proprotein convertase activity. Matrilin-3 and -4, but not matrilin-1 subunits present in matrilin-1/-3 hetero-oligomers, were identified as substrates for ADAMTS4 and ADAMTS5, whereas ADAMTS1 did not cleave any matrilin. A neo-epitope antibody raised against the N terminus of the C-terminal cleavage product of matrilin-4 detected processed matrilin-4 in cultures of primary chondrocytes as well as on cartilage sections showing that the conserved cleavage site is used in vivo. PMID:19531486

  20. Modulation of bovine microvascular endothelial cell proteolytic properties by inhibitors of angiogenesis.

    PubMed

    Pepper, M S; Vassalli, J D; Wilks, J W; Schweigerer, L; Orci, L; Montesano, R

    1994-08-01

    A tightly controlled increase in extracellular proteolysis, restricted both in time and space, is an important component of the angiogenic process, while anti-proteolysis is effective in inhibiting angiogenesis. By focussing on the plasminogen activator (PA)-plasmin system, the objective of the present studies was to assess whether previously described inhibitors of angiogenesis modify bovine microvascular endothelial cell proteolytic properties. We demonstrate that although synthetic angiostatic steroids (U-24067 and U-42129), heparin, suramin, interferon alpha-2a, and retinoic acid are all inhibitors of in vitro angiogenesis, each of these agents has distinct effects on the plasminogen-dependent proteolytic system. Specifically, angiostatic steroids and interferon alpha-2a reduce urokinase-type PA (u-PA) and PA inhibitor-1 activity, while heparin and retinoic acid increase u-PA activity. Suramin reduces cell-associated u-PA activity and greatly increases PAI-1 production at doses which induce monolayer disruption. These findings demonstrate that a spectrum of alterations in extracellular proteolysis is associated with anti-angiogenesis, and that anti-angiogenesis and anti-proteolysis are not necessarily correlated. A reduction in extracellular proteolysis would be expected to reduce invasion, whereas an increase in proteolysis might modulate the activity of inhibitory cytokines, which in turn could reduce endothelial cell proliferation and migration and inhibit angiogenesis. The spectrum of effects on different elements of the PA system observed in response to the agents assessed suggests that the role of modulations in extracellular proteolytic activity in anti-angiogenesis is likely to be varied and complex. PMID:7525617

  1. 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.

  2. Amyloid-forming peptides selected proteolytically from phage display library.

    PubMed

    Koscielska-Kasprzak, Katarzyna; Otlewski, Jacek

    2003-08-01

    We demonstrated that amyloid-forming peptides could be selected from phage-displayed library via proteolysis-based selection protocol. The library of 28-residue peptides based on a sequence of the second zinc finger domain of Zif268, and computationally designed betabetaalpha peptide, FSD-1, was presented monovalently on the surface of M13 phage. The library coupled the infectivity of phage particles to proteolytic stability of a peptide introduced into the coat protein III linker. It was designed to include variants with a strong potential to fold into betabetaalpha motif of zinc finger domains, as expected from secondary structure propensities, but with no structure stabilization via zinc ion coordination. As our primary goal was to find novel monomeric betabetaalpha peptides, the library was selected for stable domains with the assumption that folded proteins are resistant to proteolysis. After less than four rounds of proteolytic selection with trypsin, chymotrypsin, or proteinase K, we obtained a number of proteolysis-resistant phage clones containing several potential sites for proteolytic attack with the proteinases. Eight peptides showing the highest proteolysis resistance were expressed and purified in a phage-free form. When characterized, the peptides possessed proteolytic resistance largely exceeding that of the second zinc finger domain of Zif268 and FSD-1. Six of the characterized peptides formed fibrils when solubilized at high concentrations. Three of them assembled into amyloids as determined through CD measurements, Congo red and thioflavin T binding, and transmission electron microscopy. PMID:12876317

  3. 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

  4. Opportunistic proteolytic processing of carbonic anhydrase 1 from Chlamydomonas in Arabidopsis reveals a novel route for protein maturation.

    PubMed

    Juvale, Parijat S; Wagner, Ryan L; Spalding, Martin H

    2016-04-01

    Proteolytic processing of secretory proteins to yield an active form generally involves specific proteolytic cleavage of a pre-protein. Multiple specific proteases have been identified that target specific pre-protein processing sites in animals. However, characterization of site-specific proteolysis of plant pre-proteins is still evolving. In this study, we characterized proteolytic processing of Chlamydomonas periplasmic carbonic anhydrase 1 (CAH1) in Arabidopsis. CAH1 pre-protein undergoes extensive post-translational modification in the endomembrane system, including glycosylation, disulfide bond formation and proteolytic removal of a peptide 'spacer' region, resulting in a mature, heterotetrameric enzyme with two large and two small subunits. We generated a series of small-scale and large-scale modifications to the spacer and flanking regions to identify potential protease target motifs. Surprisingly, we found that the endoproteolytic removal of the spacer from the CAH1 pre-protein proceeded via an opportunistic process apparently followed by further maturation via amino and carboxy peptidases. We also discovered that the spacer itself is not required for processing, which appears to be dependent only on the number of amino acids separating two key disulfide-bond-forming cysteines. Our data suggest a novel, opportunistic route for pre-protein processing of CAH1. PMID:26917556

  5. Opportunistic proteolytic processing of carbonic anhydrase 1 from Chlamydomonas in Arabidopsis reveals a novel route for protein maturation

    PubMed Central

    Juvale, Parijat S.; Wagner, Ryan L.; Spalding, Martin H.

    2016-01-01

    Proteolytic processing of secretory proteins to yield an active form generally involves specific proteolytic cleavage of a pre-protein. Multiple specific proteases have been identified that target specific pre-protein processing sites in animals. However, characterization of site-specific proteolysis of plant pre-proteins is still evolving. In this study, we characterized proteolytic processing of Chlamydomonas periplasmic carbonic anhydrase 1 (CAH1) in Arabidopsis. CAH1 pre-protein undergoes extensive post-translational modification in the endomembrane system, including glycosylation, disulfide bond formation and proteolytic removal of a peptide ‘spacer’ region, resulting in a mature, heterotetrameric enzyme with two large and two small subunits. We generated a series of small-scale and large-scale modifications to the spacer and flanking regions to identify potential protease target motifs. Surprisingly, we found that the endoproteolytic removal of the spacer from the CAH1 pre-protein proceeded via an opportunistic process apparently followed by further maturation via amino and carboxy peptidases. We also discovered that the spacer itself is not required for processing, which appears to be dependent only on the number of amino acids separating two key disulfide-bond-forming cysteines. Our data suggest a novel, opportunistic route for pre-protein processing of CAH1. PMID:26917556

  6. 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. PMID:27421483

  7. Tracing the history of the ubiquitin proteolytic system: the pioneering article.

    PubMed

    Ciechanover, Aaron

    2009-09-11

    A series of findings made by several researchers during a two-decade period between the mid-1950s and mid-1970s raised the suspicion that the lysosome might not be the organelle that degrades the bulk of cellular proteins under basal conditions. These findings predicted the existence of a nonlysosomal, adenosine triphosphate (ATP)-dependent proteolytic system. Yet, following the initial discovery of such activity in a crude cell extract, it was a single article published in this journal [A. Ciechanover, Y. Hod, A. Hershko, A heat-stable polypeptide component of an ATP-dependent proteolytic system from reticulocytes, Biochem. Biophys. Res. Commun. 81 (1978) 1100-1105], my first study as a graduate student of Avram Hershko, that made it clear that the system that catalyzes the activity is novel and complex, and does not follow the paradigm in the field of proteolysis where a single protease typically cleaves its substrate; here at least two components were required to carry out this activity, and one of them was an unusual, small, and heat-stable protein later identified as ubiquitin. PMID:19539608

  8. PMAP: databases for analyzing proteolytic events and pathways.

    PubMed

    Igarashi, Yoshinobu; Heureux, Emily; Doctor, Kutbuddin S; Talwar, Priti; Gramatikova, Svetlana; Gramatikoff, Kosi; Zhang, Ying; Blinov, Michael; Ibragimova, Salmaz S; Boyd, Sarah; Ratnikov, Boris; Cieplak, Piotr; Godzik, Adam; Smith, Jeffrey W; Osterman, Andrei L; Eroshkin, Alexey M

    2009-01-01

    The Proteolysis MAP (PMAP, http://www.proteolysis.org) is a user-friendly website intended to aid the scientific community in reasoning about proteolytic networks and pathways. PMAP is comprised of five databases, linked together in one environment. The foundation databases, ProteaseDB and SubstrateDB, are driven by an automated annotation pipeline that generates dynamic 'Molecule Pages', rich in molecular information. PMAP also contains two community annotated databases focused on function; CutDB has information on more than 5000 proteolytic events, and ProfileDB is dedicated to information of the substrate recognition specificity of proteases. Together, the content within these four databases will ultimately feed PathwayDB, which will be comprised of known pathways whose function can be dynamically modeled in a rule-based manner, and hypothetical pathways suggested by semi-automated culling of the literature. A Protease Toolkit is also available for the analysis of proteases and proteolysis. Here, we describe how the databases of PMAP can be used to foster understanding of proteolytic pathways, and equally as significant, to reason about proteolysis. PMID:18842634

  9. PMAP: databases for analyzing proteolytic events and pathways

    PubMed Central

    Igarashi, Yoshinobu; Heureux, Emily; Doctor, Kutbuddin S.; Talwar, Priti; Gramatikova, Svetlana; Gramatikoff, Kosi; Zhang, Ying; Blinov, Michael; Ibragimova, Salmaz S.; Boyd, Sarah; Ratnikov, Boris; Cieplak, Piotr; Godzik, Adam; Smith, Jeffrey W.; Osterman, Andrei L.; Eroshkin, Alexey M.

    2009-01-01

    The Proteolysis MAP (PMAP, http://www.proteolysis.org) is a user-friendly website intended to aid the scientific community in reasoning about proteolytic networks and pathways. PMAP is comprised of five databases, linked together in one environment. The foundation databases, ProteaseDB and SubstrateDB, are driven by an automated annotation pipeline that generates dynamic ‘Molecule Pages’, rich in molecular information. PMAP also contains two community annotated databases focused on function; CutDB has information on more than 5000 proteolytic events, and ProfileDB is dedicated to information of the substrate recognition specificity of proteases. Together, the content within these four databases will ultimately feed PathwayDB, which will be comprised of known pathways whose function can be dynamically modeled in a rule-based manner, and hypothetical pathways suggested by semi-automated culling of the literature. A Protease Toolkit is also available for the analysis of proteases and proteolysis. Here, we describe how the databases of PMAP can be used to foster understanding of proteolytic pathways, and equally as significant, to reason about proteolysis. PMID:18842634

  10. 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. PMID:23489902

  11. 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. PMID:24122629

  12. Effects of Ghrelin on the Proteolytic Pathways of Alzheimer's Disease Neuronal Cells.

    PubMed

    Cecarini, Valentina; Bonfili, Laura; Cuccioloni, Massimiliano; Keller, Jeffrey N; Bruce-Keller, Annadora J; Eleuteri, Anna Maria

    2016-07-01

    Ghrelin is an orexigenic hormone with a role in the onset and progression of neurodegenerative disorders. It has been recently associated to Alzheimer's disease (AD) for its neuroprotective and anti-apoptotic activity. In the present study, we dissected the effect of ghrelin treatment on the two major intracellular proteolytic pathways, the ubiquitin-proteasome system (UPS) and autophagy, in cellular models of AD (namely SH-SY5Y neuroblastoma cells stably transfected with either the wild-type AβPP gene or the 717 valine-to-glycine AβPP-mutated gene). Ghrelin showed a growth-promoting effect on neuronal cells inducing also time-dependent modifications of the growth hormone secretagogue receptor type 1 (GHS-R1) expression. Interestingly, we demonstrated for the first time that ghrelin was able to activate the proteasome in neural cells playing also a role in the interplay between the UPS and autophagy. Our data provide a novel mechanism by which circulating hormones control neural homeostasis through the regulation of proteolytic pathways implicated in AD. PMID:26033219

  13. Involvement of activation of PI3K/Akt pathway in the protective effects of puerarin against MPP+-induced human neuroblastoma SH-SY5Y cell death.

    PubMed

    Zhu, Guoqi; Wang, Xuncui; Wu, Shengbing; Li, Qinglin

    2012-03-01

    In an attempt to clarify the protective effect of puerarin on toxin-insulted dopaminergic neuronal death, this present study was carried out by using a typical Parkinson's disease (PD) model - 1-methyl-4-phenylpyridinium iodide (MPP(+))-induced dopaminergic SH-SY5Y cellular model. Data are presented, which showed that puerarin up-regulated Akt phosphorylation in both of MPP(+)-treated and non-MPP(+)-treated cells. The presence of PI3K inhibitor LY294002 completely blocked puerarin-induced activation of Akt phosphorylation. Moreover, puerarin decreased MPP(+)-induced cell death, which was blocked by phosphoinositide 3-kinase (PI3K) inhibitor LY294002. We further demonstrated that puerarin protected against MPP(+)-induced p53 nuclear accumulation, Puma (p53-upregulated mediator of apoptosis) and Bax expression and caspase-3-dependent programmed cell death (PCD). This protection was blocked by applying a PI3K/Akt inhibitor. Additionally, it was Pifithrin-α, but not Pifithrin-μ, which blocked MPP(+)-induced Puma and Bax expression, caspase-3 activation and cell death. Collectively, these data suggest that the activation of PI3K/Akt pathway is involved in the protective effect of puerarin against MPP(+)-induced neuroblastoma SH-SY5Y cell death through inhibiting nuclear p53 accumulation and subsequently caspase-3-dependent PCD. Puerarin might be a potential therapeutic agent for PD. PMID:22265823

  14. 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

  15. Proteolytic yeasts isolated from raw, ripe tomatoes and metabiotic association of Geotrichum candidum with Salmonella.

    PubMed

    Wade, Wendy N; Vasdinnyei, Rita; Deak, Tibor; Beuchat, Larry R

    2003-09-01

    Metabiotic associations between food-borne fungi and bacteria capable of causing human diseases are a public health concern. A survey of decayed and damaged, uncooked, ripe tomatoes was done to determine the presence and prevalence of yeasts capable of increasing the pH pulp tissue, thus creating a more favorable environment for survival and growth of enteric pathogens. Sixty-two of the 371 (16.7%) fungi isolated from 215 decayed or damaged tomatoes; 12 of the 62 (19.4%) yeasts showed proteolytic activity on gelatin agar (GA) and/or standard methods caseinate (SMC) agar. The pH of tomato pericarp (pulp) tissue from which 9 of the 12 yeasts were isolated ranged from 4.3 to 7.5 (mean=5.3) compared to 4.2-5.1 (mean=4.8) for sound pulp tissue in the same tomatoes. The 12 proteolytic yeasts consisted of four strains of Cryptococcus albidus, two strains each of Debaryomyces hansenii and Trichosporon pullulans, and one strain each of Cryptococcus humicolus, Cryptococcus laurentii, Geotrichum candidum, and Sporidiobolus pararoseus. Survival and growth characteristics of a five-serotype mixture of Salmonella co-inoculated with G. candidum into sound (not chill injured) and chill-injured tomatoes were studied. Storage of sound tomatoes at 15 degrees C for 10 days resulted in an increase in population of 7.6 log(10) cfu of Salmonella/g of a 2-g sample of co-infected pulp tissue. Increases were less in tissue inoculated with Salmonella only, Salmonella on day 0 followed by G. candidum on day 3, or G. candidum on day 0 followed by Salmonella on day 3. Trends were similar in sound inoculated tomatoes stored at 25 degrees C. Growth of Salmonella was enhanced in chill-injured tomatoes compared to sound tomatoes; a population of 10 log(10) cfu/g of chill-injured pulp tissue was reached within 10 days at 25 degrees C. Results clearly show that growth of a proteolytic, alkalinizing yeast such as G. candidum in raw tomatoes enhances conditions for growth of Salmonella. The removal of

  16. Calmodulin inhibitors trigger the proteolytic processing of membrane type-1 matrix metalloproteinase, but not its shedding in glioblastoma cells.

    PubMed Central

    Annabi, B; Pilorget, A; Bousquet-Gagnon, N; Gingras, D; Béliveau, R

    2001-01-01

    Most transmembrane proteins are subjected to limited proteolysis by cellular proteases, and stimulation of cleavage of membrane proteins by calmodulin (CaM) inhibitors was recently shown. The present study investigated the ability of several CaM inhibitors to induce the proteolytic cleavage of the membrane type-1 matrix metalloproteinase (MT1-MMP) from the cell surface of highly invasive U-87 glioblastoma cells. Although no shedding of a soluble MT1-MMP form was induced by CaM inhibitors in the conditioned media, we showed that these inhibitors induced MT1-MMP proteolytic processing to the 43 kDa membrane-bound inactive form that was not correlated with an increase in proMMP-2 activation but rather with an increase in tissue inhibitor of MMPs (TIMP)-2 expression levels. Moreover, this proteolytic processing was sensitive to marimastat suggesting the involvement of MMPs. Interestingly, CaM inhibitors antagonized concanavalin A- and cytochalasin D-induced proMMP-2 activation, and affected the cytoskeletal actin organization resulting in the loss of migratory potential of U-87 glioblastoma cells. Cytoplasmic tail-truncated MT1-MMP constructs expressed in COS-7 cells were also affected by CaM inhibitors suggesting that these inhibitors stimulated MT1-MMP proteolytic processing by mechanisms independent of the CaM-substrate interaction. We also propose that TIMP-2 acts as a negative regulator of MT1-MMP-dependent activities promoted by the action of CaM inhibitors in U-87 glioblastoma cells. PMID:11583578

  17. 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

  18. Interaction specificity between the chaperone and proteolytic components of the cyanobacterial Clp protease.

    PubMed

    Tryggvesson, Anders; Ståhlberg, Frida M; Mogk, Axel; Zeth, Kornelius; Clarke, Adrian K

    2012-09-01

    The Clp protease is conserved among eubacteria and most eukaryotes, and uses ATP to drive protein substrate unfolding and translocation into a chamber of sequestered proteolytic active sites. In plant chloroplasts and cyanobacteria, the essential constitutive Clp protease consists of the Hsp100/ClpC chaperone partnering a proteolytic core of catalytic ClpP and noncatalytic ClpR subunits. In the present study, we have examined putative determinants conferring the highly specific association between ClpC and the ClpP3/R core from the model cyanobacterium Synechococcus elongatus. Two conserved sequences in the N-terminus of ClpR (tyrosine and proline motifs) and one in the N-terminus of ClpP3 (MPIG motif) were identified as being crucial for the ClpC-ClpP3/R association. These N-terminal domains also influence the stability of the ClpP3/R core complex itself. A unique C-terminal sequence was also found in plant and cyanobacterial ClpC orthologues just downstream of the P-loop region previously shown in Escherichia coli to be important for Hsp100 association to ClpP. This R motif in Synechococcus ClpC confers specificity for the ClpP3/R core and prevents association with E. coli ClpP; its removal from ClpC reverses this core specificity. PMID:22657732

  19. Structural identification and proteolytic effects of the hatching enzyme from starfish Asterias amurensis.

    PubMed

    Li, Zhi Jiang; Kim, Sang Moo

    2014-07-01

    Hatching enzyme (HE) is secreted from the blastula stage during fertilization and can cleave the egg membrane. The structural identification and proteolytic effects on the collagen and fibrinogen were investigated in this study. Approximate 20 kDa of Asn-linked oligosaccharides were attached to the HE. Five peptide fragments of the starfish HE were homogenous to those of the coat matrix protein of starfish Patiria pectinifera. Amino acids of the starfish HE consisted of mainly Leu (10.0%), Asp (12.5%), and Glu (12.8%). Collagenolytic and fibrinolytic activities of the starfish HE were weaker than those of collagenase and α-chymotrypsin. The degree values of hydrolysis for collagenase and α- chymotrypsin were significantly higher than those of HE in a dose- and time-dependent manner. The peptide mappings of the starfish HE on the collagenolysis (110.7, 84.7, and 20.8 kDa) and fibrinogenolysis (34, 30, and 29 kDa) were different from those of collagenase and α-chymotrypsin. Based on the proteolytic effects on the collagen and fibrinogen, the starfish hatching enzyme might have the potential application to remove the matrix composition in scar or keloid tissue. PMID:24559163

  20. Functionalized Biopolymer Particles Enhance Performance of a Tissue-Protective Peptide under Proteolytic and Thermal Stress.

    PubMed

    Dooley, Kevin; Devalliere, Julie; Uygun, Basak E; Yarmush, Martin L

    2016-06-13

    Cutaneous burns are often exacerbated by poor perfusion and subsequent necrosis of the microvasculature surrounding the primary injury. Preservation of these vessels can reduce necrotic tissue expansion and increase success rates of skin graft procedures. Recent work has identified a peptide derived from erythropoietin, ARA290, with the ability to mediate tissue protection in a variety of cell types. Here we demonstrate the advantages of fusing ARA290 to an elastin-like polypeptide (ELP) to salvage microvascular endothelial cells in harsh proteolytic conditions following thermal shock. These fusion proteins were expressed recombinantly in bacterial hosts and rapidly purified by inverse transition cycling. They were shown to spontaneously aggregate into particles at subphysiological temperatures. The bifunctional submicron particles were resistant to digestion in enzymes upregulated after burn injury. Furthermore, the data strongly suggest these ARA290-functionalized particles were superior to treatment with the peptide alone in preventing microvascular cell death in these conditions. The results bring to light an efficient and cost-effective strategy for the delivery therapeutic peptides to proteolytically active wound sites. PMID:27219509

  1. Nigerian bonny-light crude oil induces alteration in testicular stress response proteins and caspase-3 dependent apoptosis in albino wistar rats.

    PubMed

    Ebokaiwe, Azubuike P; D'Cruz, Cynthia S; Jubendradass, R; Amala Rani, Judith S; Mathur, Premendu P; Farombi, Ebenezer O

    2015-02-01

    In the past few decades, there has been much concern about the adverse health effects of environmental contaminants in general and Crude Oil in particular around the Niger Delta region of Nigeria where all the crude Oil exploration is taking place. Studies have shown the repro-toxic effects of Bonny-light crude oil (BLCO). However, the insight into the mechanisms of gonadal toxicity induced by BLCO is not well known. In this study, we sought to elucidate the mechanism(s) underpinning the gonadal effects within hours of exposure to BLCO. Experimental rats were divided into five groups of four each. Animals were orally administered with a single dose of BLCO (800 mg/kg body weight) and killed at 0, 6, 12, 24, and 72 h post-treatment. The levels and time-course of induction of stress response proteins and apoptosis-related proteins like cytochorome C, caspase 3 and procaspase 9, Fas-FasL, NF-kB and TNF-α were determined to assess sequential induction of apoptosis in the rat testis. DNA damage was assessed by TUNEL assay. Administration of BLCO resulted in a significant increase in the levels of stress response proteins and apoptotis- related proteins as early as 6 h following exposure. Time-dependent elevations in the levels of the proteins were observed. The DNA damage was measured and showed time-dependent increase in the TUNEL positive cells of testicular cells. The study demonstrates induction of testicular apoptosis in adult rats following exposure to a single dose of BLCO. PMID:24106129

  2. Degradation of oxidized proteins by the proteasome: Distinguishing between the 20S, 26S, and immunoproteasome proteolytic pathways.

    PubMed

    Raynes, Rachel; Pomatto, Laura C D; Davies, Kelvin J A

    2016-08-01

    The proteasome is a ubiquitous and highly plastic multi-subunit protease with multi-catalytic activity that is conserved in all eukaryotes. The most widely known function of the proteasome is protein degradation through the 26S ubiquitin-proteasome system, responsible for the vast majority of protein degradation during homeostasis. However, the proteasome also plays an important role in adaptive immune responses and adaptation to oxidative stress. The unbound 20S proteasome, the core common to all proteasome conformations, is the main protease responsible for degrading oxidized proteins. During periods of acute stress, the 19S regulatory cap of the 26S proteasome disassociates from the proteolytic core, allowing for immediate ATP/ubiquitin-independent protein degradation by the 20S proteasome. Despite the abundance of unbound 20S proteasome compared to other proteasomal conformations, many publications fail to distinguish between the two proteolytic systems and often regard the 26S proteasome as the dominant protease. Further confounding the issue are the differential roles these two proteolytic systems have in adaptation and aging. In this review, we will summarize the increasing evidence that the 20S core proteasome constitutes the major conformation of the proteasome system and that it is far from a latent protease requiring activation by binding regulators. PMID:27155164

  3. Assembly and proteolytic processing of mycobacterial ClpP1 and ClpP2

    PubMed Central

    2011-01-01

    Background Caseinolytic proteases (ClpPs) are barrel-shaped self-compartmentalized peptidases involved in eliminating damaged or short-lived regulatory proteins. The Mycobacterium tuberculosis (MTB) genome contains two genes coding for putative ClpPs, ClpP1 and ClpP2 respectively, that are likely to play a role in the virulence of the bacterium. Results We report the first biochemical characterization of ClpP1 and ClpP2 peptidases from MTB. Both proteins were produced and purified in Escherichia coli. Use of fluorogenic model peptides of diverse specificities failed to show peptidase activity with recombinant mycobacterial ClpP1 or ClpP2. However, we found that ClpP1 had a proteolytic activity responsible for its own cleavage after the Arg8 residue and cleavage of ClpP2 after the Ala12 residue. In addition, we showed that the absence of any peptidase activity toward model peptides was not due to an obstruction of the entry pore by the N-terminal flexible extremity of the proteins, nor to an absolute requirement for the ClpX or ClpC ATPase complex. Finally, we also found that removing the putative propeptides of ClpP1 and ClpP2 did not result in cleavage of model peptides. We have also shown that recombinant ClpP1 and ClpP2 do not assemble in the conventional functional tetradecameric form but in lower order oligomeric species ranging from monomers to heptamers. The concomitant presence of both ClpP1 and ClpP2 did not result in tetradecameric assembly. Deleting the amino-terminal extremity of ClpP1 and ClpP2 (the putative propeptide or entry gate) promoted the assembly in higher order oligomeric species, suggesting that the flexible N-terminal extremity of mycobacterial ClpPs participated in the destabilization of interaction between heptamers. Conclusion Despite the conservation of a Ser protease catalytic triad in their primary sequences, mycobacterial ClpP1 and ClpP2 do not have conventional peptidase activity toward peptide models and display an unusual

  4. Antioxidative and proteolytic systems protect mitochondria from oxidative damage in S-deficient Arabidopsis thaliana.

    PubMed

    Ostaszewska-Bugajska, Monika; Rychter, Anna M; Juszczuk, Izabela M

    2015-08-15

    We examined the functioning of the antioxidative defense system in Arabidopsis thaliana under sulphur (S) deficiency with an emphasis on the role of mitochondria. In tissue extracts and in isolated mitochondria from S-deficient plants, the concentration of non-protein thiols declined but protein thiols did not change. Superoxide anion and hydrogen peroxide were accumulated in leaf blades and the generation of superoxide anion by isolated mitochondria was higher. Lower abundance of reduced (GSH) plus oxidized (GSSG) glutathione in the leaf and root tissues, and leaf mitochondria from S-deficient plants was accompanied by a decrease in the level of GSH and the changes in the GSH/GSSG ratios. In the chloroplasts, the total level of glutathione decreased. Lower levels of reduced (AsA) and oxidized (DHA) ascorbate were reflected in much higher ratios of AsA/DHA. Sulphur deficiency led to an increase in the activity of cytosolic, mitochondrial and chloroplastic antioxidative enzymes, peroxidases, catalases and superoxide dismutases. The protein carbonyl level was higher in the leaves of S-deficient plants and in the chloroplasts, while in the roots, leaf and root mitochondria it remained unchanged. Protease activity in leaf extracts of S-deficient plants was higher, but in root extracts it did not differ. The proteolytic system reflected subcellular specificity. In leaf and root mitochondria the protease activity was higher, whereas in the chloroplasts it did not change. We propose that the preferential incorporation of S to protein thiols and activation of antioxidative and proteolytic systems are likely important for the survival of S-deficient plants and that the mitochondria maintain redox homeostasis. PMID:26339750

  5. Comparison of Proliferative Effect of Human Lactoferrin and Its Proteolytic Peptide on Normal and Transformed Epithelial Cells.

    PubMed

    Hwang, Sae-Mi; Chung, Il Yup; Jo, Jae-Hyung; Yoon, Tae-Joong; Lee, Hyune-Hwan

    2016-01-01

    Human lactoferrin (hLF) is an iron-binding glycoprotein with a variety of functions. hLF undergoes proteolytic cleavage to smaller peptides in the stomach following ingestion. In the present study, we evaluated the effects of hLF and its proteolytic product, human lactoferrin peptide (hLFP), on the proliferation of two epithelial cells, HEK293 normal cells and KATO III gastric carcinoma cells, using an MTT assay and expression of proliferative nuclear cell antigen (PCNA), a notable proliferation marker. When the two epithelial cells were stimulated with hLF and hLFP in the presence of fetal bovine serum (FBS), hLFP stimulated proliferation of both cell types at lower concentrations than hLF by two orders of magnitude. The cancer cells exhibited proliferative responses to both hLF and hLFP at lower concentrations by 2∼3 orders of magnitude than the normal cells. Either hLF or hLFP alone did not support appreciable proliferation of these cell lines in the absence or low concentrations of FBS. Bovine serum albumin or its proteolytic product failed to promote cellular proliferation even in the presence of 10 % FBS, indicating the specificity of the proliferative activity of hLF and hLFP. These data highlight feasibility of hLF and its peptide for adjuvants for tissue culture medium. PMID:26400493

  6. The role of proteolytic enzymes in degradation of plant tissues: Summary report

    SciTech Connect

    Lewosz, J.; Kelman, A.; Sequeira, L.

    1989-01-01

    The proteolytic enzymes produced by Erwinia carotovora subsp. carotovora (Ecc-strain SR 394) grown on various media were examined by isoelectrofocusing in polyacrylamide gels over a pH range of 3-10. In addition to the main protease present in culture filtrates, low concentrations of several other proteases were present in extracts from potato tubers infected by Ecc. Proteases from all these sources were similar and had the following properties: pH optimum near 8.0, calcium dependent, insensitive to serine proteinase and SH-proteinase inhibitors, inhibited by EDTA, and highly thermostable. These enzymes degraded gelatin, soluble collagen and Hide Powder Azure, and showed weak activity on casein, but did not degrade insoluble collagen or elastin.

  7. Tumor-Specific Proteolytic Processing of Cyclin E Generates Hyperactive Lower-Molecular-Weight Forms

    PubMed Central

    Porter, Donald C.; Zhang, Ning; Danes, Christopher; McGahren, Mollianne J.; Harwell, Richard M.; Faruki, Shamsa; Keyomarsi, Khandan

    2001-01-01

    Cyclin E is a G1 cyclin essential for S-phase entry and has a profound role in oncogenesis. Previously this laboratory found that cyclin E is overexpressed and present in lower-molecular-weight (LMW) isoforms in breast cancer cells and tumor tissues compared to normal cells and tissues. Such alteration of cyclin E is linked to poor patient outcome. Here we report that the LMW forms of cyclin E are hyperactive biochemically and they can more readily induce G1-to-S progression in transfected normal cells than the full-length form of the protein can. Through biochemical and mutational analyses we have identified two proteolytically sensitive sites in the amino terminus of human cyclin E that are cleaved to generate the LMW isoforms found in tumor cells. Not only are the LMW forms of cyclin E functional, as they phosphorylate substrates such as histone H1 and GST-Rb, but also their activities are higher than the full-length cyclin E. These nuclear localized LMW forms of cyclin E are also biologically functional, as their overexpression in normal cells increases the ability of these cells to enter S and G2/M. Lastly, we show that cyclin E is selectively cleaved in vitro by the elastase class of serine proteases to generate LMW forms similar to those observed in tumor cells. These studies suggest that the defective entry into and exit from S phase by tumor cells is in part due to the proteolytic processing of cyclin E, which generates hyperactive LMW isoforms whose activities have been modified from that of the full-length protein. PMID:11509668

  8. A Familial Mutation Renders Atrial Natriuretic Peptide Resistant to Proteolytic Degradation*

    PubMed Central

    Dickey, Deborah M.; Yoder, Andrea R.; Potter, Lincoln R.

    2009-01-01

    A heterozygous frameshift mutation causing a 12-amino acid extension to the C terminus of atrial natriuretic peptide (ANP) was recently genetically linked to patients with familial atrial fibrillation (Hodgson-Zingman, D. M., Karst, M. L., Zingman, L. V., Heublein, D. M., Darbar, D., Herron, K. J., Ballew, J. D., de Andrade, M., Burnett, J. C., Jr., and Olson, T. M. (2008) N. Engl. J. Med. 359, 158–165). The frameshift product (fsANP), but not wild-type ANP (wtANP), was elevated in the serum of affected patients, but the molecular basis for the elevated peptide concentrations was not determined. Here, we measured the ability of fsANP to interact with natriuretic peptide receptors and to be proteolytically degraded. fsANP and wtANP bound and activated human NPR-A and NPR-C similarly, whereas fsANP had a slightly increased efficacy for human NPR-B. Proteolytic susceptibility was addressed with novel bioassays that measure the time required for kidney membranes or purified neutral endopeptidase to abolish ANP-dependent activation of NPR-A. The half-life of fsANP was markedly greater than that of wtANP in both assays. Additional membrane proteolysis studies indicated that wtANP and fsANP are preferentially degraded by neutral endopeptidase and serine peptidases, respectively. These data indicate that the familial ANP mutation associated with atrial fibrillation has only minor effects on natriuretic peptide receptor interactions but markedly modifies peptide proteolysis. PMID:19458086

  9. Wound dressings for a proteolytic-rich environment.

    PubMed

    Vasconcelos, Andreia; Cavaco-Paulo, Artur

    2011-04-01

    Wound dressings have experienced continuous and significant changes over the years based on the knowledge of the biochemical events associated with chronic wounds. The development goes from natural materials used to just cover and conceal the wound to interactive materials that can facilitate the healing process, addressing specific issues in non-healing wounds. These new types of dressings often relate with the proteolytic wound environment and the bacteria load to enhance the healing. Recently, the wound dressing research is focusing on the replacement of synthetic polymers by natural protein materials to delivery bioactive agents to the wounds. This article provides an overview on the novel protein-based wound dressings such as silk fibroin keratin and elastin. The improved properties of these dressings, like the release of antibiotics and growth factors, are discussed. The different types of wounds and the effective parameters of healing process will be reviewed. PMID:21360151

  10. The fine-tuning of proteolytic pathways in Alzheimer's disease.

    PubMed

    Cecarini, Valentina; Bonfili, Laura; Cuccioloni, Massimiliano; Mozzicafreddo, Matteo; Angeletti, Mauro; Keller, Jeffrey N; Eleuteri, Anna Maria

    2016-09-01

    Several integrated proteolytic systems contribute to the maintenance of cellular homeostasis through the continuous removal of misfolded, aggregated or oxidized proteins and damaged organelles. Among these systems, the proteasome and autophagy play the major role in protein quality control, which is a fundamental issue in non-proliferative cells such as neurons. Disturbances in the functionality of these two pathways are frequently observed in neurodegenerative diseases, like Alzheimer's disease, and reflect the accumulation of protease-resistant, deleterious protein aggregates. In this review, we explored the sophisticated crosstalk between the ubiquitin-proteasome system and autophagy in the removal of the harmful structures that characterize Alzheimer's disease neurons. We also dissected the role of the numerous shuttle factors and chaperones that, directly or indirectly interacting with ubiquitin and LC3, are used for cargo selection and delivery to one pathway or the other. PMID:27120560

  11. Four proteolytic processes of myocardium, one insensitive to thiol reactive agents and thiol protease inhibitor.

    PubMed

    Thorne, D P; Lockwood, T D

    1993-07-01

    Four distinct processes mediating protein degradation were identified in the Langendorff perfused rat heart. Hearts were biosynthetically labeled in vitro with [3H]leucine for 10 min. The subsequent release of [3H]leucine at 1.5-min intervals (2 mM nonradioactive leucine) was determined from 20 min to 8 h after labeling in rhythmically contracting hearts. Rapid turnover proteins were eliminated during the first 3 h; this degradation was not inhibited by insulin (5 nM) or isoproterenol (0.5 microM). However, the nontoxic thiol reactive agent diamide (100 microM) caused a complete inhibition of the [3H]leucine release from rapidly degraded proteins. After the elimination of rapidly degraded proteins at 3 h, the release of [3H]leucine was inhibited 35-40% by insulin (5 nM) or the lysosomal inhibitor chloroquine (30 microM), thereby defining a second vesicular process. The beta-agonist isoproterenol (0.5 microM) or the nonselective alpha-agonist naphazoline (100 microM) caused 30-35% proteolytic inhibitions, defining a third adrenergic-responsive process. The inhibitory effects of simultaneously combined insulin and chloroquine did not exceed the effect of either agent alone. However, the combined effects of insulin and isoproterenol were additive, inhibiting two-thirds of basal degradation. Beginning at 3 h after labeling a 75% proteolytic inhibition resulted from the thiol reactive agents diamide (100 microM) or N-ethylmaleimide (10 microM); the thiol protease active site inhibitor trans-epoxysuccinly-L-leucylamino-(4-quinidino)butane (50 microM) caused 65% inhibition. The 75% inhibition caused by diamide includes both the insulin-responsive and beta-adrenergic-responsive pathways. A novel fourth proteolytic process (25% of proteolysis) was thereby distinguished from the above three by its resistance to inhibition by insulin, adrenergic agonists, thiol reactive agents, or thiol protease inhibitor. Only the adrenergic-responsive process was correlated with changes in

  12. Kallikrein-8 Proteolytically Processes Human Papillomaviruses in the Extracellular Space To Facilitate Entry into Host Cells

    PubMed Central

    Cerqueira, Carla; Samperio Ventayol, Pilar; Vogeley, Christian

    2015-01-01

    ABSTRACT The entry of human papillomaviruses into host cells is a complex process. It involves conformational changes at the cell surface, receptor switching, internalization by a novel endocytic mechanism, uncoating in endosomes, trafficking of a subviral complex to the Golgi complex, and nuclear entry during mitosis. Here, we addressed how the stabilizing contacts in the capsid of human papillomavirus 16 (HPV16) may be reversed to allow uncoating of the viral genome. Using biochemical and cell-biological analyses, we determined that the major capsid protein L1 underwent proteolytic cleavage during entry. In addition to a dispensable cathepsin-mediated proteolysis that occurred likely after removal of capsomers from the subviral complex in endosomes, at least two further proteolytic cleavages of L1 were observed, one of which was independent of the low-pH environment of endosomes. This cleavage occurred extracellularly. Further analysis showed that the responsible protease was the secreted trypsin-like serine protease kallikrein-8 (KLK8) involved in epidermal homeostasis and wound healing. Required for infection, the cleavage was facilitated by prior interaction of viral particles with heparan sulfate proteoglycans. KLK8-mediated cleavage was crucial for further conformational changes exposing an important epitope of the minor capsid protein L2. Occurring independently of cyclophilins and of furin that mediate L2 exposure, KLK8-mediated cleavage of L1 likely facilitated access to L2, located in the capsid lumen, and potentially uncoating. Since HPV6 and HPV18 also required KLK8 for entry, we propose that the KLK8-dependent entry step is conserved. IMPORTANCE Our analysis of the proteolytic processing of incoming HPV16, an etiological agent of cervical cancer, demonstrated that the capsid is cleaved extracellularly by a serine protease active during wound healing and that this cleavage was crucial for infection. The cleavage of L1 is one of at least four structural

  13. Proteolytic Cleavage of Apolipoprotein E in the Down Syndrome Brain

    PubMed Central

    Day, Ryan J.; McCarty, Katie L.; Ockerse, Kayla E.; Head, Elizabeth; Rohn, Troy T.

    2016-01-01

    Down syndrome (DS) is one of the most common genetic causes of intellectual disability and is characterized by a number of behavioral as well as cognitive symptoms. Many of the neuropathological features of early-onset Alzheimer’s disease (AD) including senile plaques and neurofibrillary tangles (NFTs) are also present in people with DS as a result of triplication of the amyloid precursor gene on chromosome 21. Evidence suggests that harboring one or both apolipoprotein E4 (APOE4) alleles may increase the risk for AD due to the proteolytic cleavage of apoE4 and a subsequent loss of function. To investigate a role for the apoE proteolysis in vivo, we compared three autopsy groups; 7 DS with AD neuropathology cases over 40 years, 5 young DS cases without AD pathology under 40 years (YDS) and 5 age-matched control cases over 40 years by immunohistochemistry utilizing an antibody that detects the amino-terminal fragment of apoE. Application of this antibody, termed the amino-terminal apoE fragment antibody (nApoECF) revealed labeling of pyramidal neurons in the frontal cortex of YDS cases, whereas in the DS-AD group, labeling with nApoECF was prominent within NFTs. NFT labeling with nApoECF was significantly greater in the hippocampus versus the frontal cortex in the same DS-AD cases, suggesting a regional distribution of truncated apoE. Colocalization immunofluorescence experiments indicated that 52.5% and 53.2% of AT8- and PHF-1-positive NFTs, respectively, also contained nApoECF. Collectively, these data support a role for the proteolytic cleavage of apoE in DS and suggest that apoE fragmentation is closely associated with NFTs. PMID:27330841

  14. Proteolytic Cleavage of Apolipoprotein E in the Down Syndrome Brain.

    PubMed

    Day, Ryan J; McCarty, Katie L; Ockerse, Kayla E; Head, Elizabeth; Rohn, Troy T

    2016-05-01

    Down syndrome (DS) is one of the most common genetic causes of intellectual disability and is characterized by a number of behavioral as well as cognitive symptoms. Many of the neuropathological features of early-onset Alzheimer's disease (AD) including senile plaques and neurofibrillary tangles (NFTs) are also present in people with DS as a result of triplication of the amyloid precursor gene on chromosome 21. Evidence suggests that harboring one or both apolipoprotein E4 (APOE4) alleles may increase the risk for AD due to the proteolytic cleavage of apoE4 and a subsequent loss of function. To investigate a role for the apoE proteolysis in vivo, we compared three autopsy groups; 7 DS with AD neuropathology cases over 40 years, 5 young DS cases without AD pathology under 40 years (YDS) and 5 age-matched control cases over 40 years by immunohistochemistry utilizing an antibody that detects the amino-terminal fragment of apoE. Application of this antibody, termed the amino-terminal apoE fragment antibody (nApoECF) revealed labeling of pyramidal neurons in the frontal cortex of YDS cases, whereas in the DS-AD group, labeling with nApoECF was prominent within NFTs. NFT labeling with nApoECF was significantly greater in the hippocampus versus the frontal cortex in the same DS-AD cases, suggesting a regional distribution of truncated apoE. Colocalization immunofluorescence experiments indicated that 52.5% and 53.2% of AT8- and PHF-1-positive NFTs, respectively, also contained nApoECF. Collectively, these data support a role for the proteolytic cleavage of apoE in DS and suggest that apoE fragmentation is closely associated with NFTs. PMID:27330841

  15. TRPV1 Activation in Primary Cortical Neurons Induces Calcium-Dependent Programmed Cell Death.

    PubMed

    Song, Juhyun; Lee, Jun Hong; Lee, Sung Ho; Park, Kyung Ah; Lee, Won Taek; Lee, Jong Eun

    2013-03-01

    Transient receptor potential cation channel, subfamily V, member 1 (TRPV1, also known as vanilloid receptor 1) is a receptor that detects capsaicin, a pungent component of chili peppers, and noxious heat. Although its function in the primary nociceptor as a pain receptor is well established, whether TRPV1 is expressed in the brain is still under debate. In this study, the responses of primary cortical neurons were investigated. Here, we report that 1) capsaicin induces caspase-3-dependent programmed cell death, which coincides with increased production of nitric oxide and peroxynitrite ; that 2) the prolonged capsaicin treatment induces a steady increase in the degree of capase-3 activation, which is prevented by the removal of capsaicin; 3) and that blocking calcium entry and calcium-mediated signaling prevents capsaicin-induced cell death. These results indicate that cortical neurons express TRPV1 whose prolonged activation causes cell death. PMID:23585723

  16. Thrombin induces endothelial cell growth via both a proteolytic and a non-proteolytic pathway.

    PubMed Central

    Herbert, J M; Dupuy, E; Laplace, M C; Zini, J M; Bar Shavit, R; Tobelem, G

    1994-01-01

    Binding of 125I-thrombin to human umbilical vein endothelial cells (HUVECs) was specifically displaced by the synthetic tetradecapeptide SFLLRNPNDKYEPF, named thrombin receptor agonist peptide (TRAP), which has recently been described as a peptide mimicking the new N-terminus created by cleavage of the thrombin receptor, and F-14, a tetradecapeptide representing residues 365-378 of the human alpha-thrombin B chain. Binding of 125I-TRAP to HUVECs was time-dependent, reversible and saturable, showing high affinity (KD = 1.5 +/- 0.4 microM) and high binding capacity (Bmax. = 7.1 +/- 0.6 x 10(6) sites/cell) (n = 3). Unlabelled thrombin and TRAP competitively and selectively inhibited the specific binding of 125I-TRAP with IC50 values of 5.8 +/- 0.7 nM and 2.8 +/- 0.4 microM respectively, whereas F-14 remained ineffective at displacing 125I-TRAP from its binding sites, suggesting the presence of at least two different types of thrombin-binding sites on HUVECs. TRAP was a potent mitogen for HUVECs in culture. Both TRAP and alpha-thrombin stimulated the proliferation of HUVECs with half-maximum mitogenic responses between 1 and 10 nM. F-14 also promoted HUVEC growth. The mitogenic effects of F-14 and TRAP were additive. N alpha-(2-Naphthylsulphonylglycyl)-DL-p-amidinophenylalanylpiper idine (NAPAP) and hirudin (two specific inhibitors of the enzyme activity of thrombin) specifically inhibited thrombin-induced HUVEC growth (IC50 values 400 +/- 60 and 52 +/- 8 nM respectively) but remained without effect on the mitogenic effect of TRAP or F-14. This demonstrated that the mitogenic effect of alpha-thrombin for HUVECs was intimately linked to its esterolytic activity but also showed that thrombin can stimulate HUVEC growth via another non-enzymic pathway. This hypothesis was further reinforced by the fact that F-14-induced proliferation of HUVECs remained unaltered by two antibodies directed against TRAP or the cleavage site on the extracellular portion of the thrombin

  17. Proteolytic and non-proteolytic regulation of collective cell invasion: tuning by ECM density and organization

    PubMed Central

    Kumar, Sandeep; Kapoor, Aastha; Desai, Sejal; Inamdar, Mandar M.; Sen, Shamik

    2016-01-01

    Cancer cells manoeuvre through extracellular matrices (ECMs) using different invasion modes, including single cell and collective cell invasion. These modes rely on MMP-driven ECM proteolysis to make space for cells to move. How cancer-associated alterations in ECM influence the mode of invasion remains unclear. Further, the sensitivity of the two invasion modes to MMP dynamics remains unexplored. In this paper, we address these open questions using a multiscale hybrid computational model combining ECM density-dependent MMP secretion, MMP diffusion, ECM degradation by MMP and active cell motility. Our results demonstrate that in randomly aligned matrices, collective cell invasion is more efficient than single cell invasion. Although increase in MMP secretion rate enhances invasiveness independent of cell–cell adhesion, sustenance of collective invasion in dense matrices requires high MMP secretion rates. However, matrix alignment can sustain both single cell and collective cell invasion even without ECM proteolysis. Similar to our in-silico observations, increase in ECM density and MMP inhibition reduced migration of MCF-7 cells embedded in sandwich gels. Together, our results indicate that apart from cell intrinsic factors (i.e., high cell–cell adhesion and MMP secretion rates), ECM density and organization represent two important extrinsic parameters that govern collective cell invasion and invasion plasticity. PMID:26832069

  18. A region within a lumenal loop of Saccharomyces cerevisiae Ycf1p directs proteolytic processing and substrate specificity.

    PubMed

    Mason, Deborah L; Mallampalli, Monica P; Huyer, Gregory; Michaelis, Susan

    2003-06-01

    Ycf1p, a member of the yeast multidrug resistance-associated protein (MRP) subfamily of ATP-binding cassette proteins, is a vacuolar membrane transporter that confers resistance to a variety of toxic substances such as cadmium and arsenite. Ycf1p undergoes a PEP4-dependent processing event to yield N- and C-terminal cleavage products that remain associated with one another. In the present study, we sought to determine whether proteolytic cleavage is required for Ycf1p activity. We have identified a unique region within lumenal loop 6 of Ycf1p, designated the loop 6 insertion (L6(ins)), which appears to be necessary and sufficient for proteolytic cleavage, since L6(ins) can promote processing when moved to new locations in Ycf1p or into a related transporter, Bpt1p. Surprisingly, mutational results indicate that proteolytic processing is not essential for Ycf1p transport activity. Instead, the L6(ins) appears to regulate substrate specificity of Ycf1p, since certain mutations in this region lower cellular cadmium resistance with a concomitant gain in arsenite resistance. Although some of these L6(ins) mutations block processing, there is no correlation between processing and substrate specificity. The activity profiles of the Ycf1p L6(ins) mutants are dramatically affected by the strain background in which they are expressed, raising the possibility that another cellular component may functionally impact Ycf1p activity. A candidate component may be a new full-length MRP-type transporter (NFT1), reported in the Saccharomyces Genome Database as two adjacent open reading frames, YKR103w and YKR104w, but which we show here is present in most Saccharomyces strains as a single open reading frame. PMID:12796304

  19. The leader proteinase of foot-and-mouth disease virus: structure-function relationships in a proteolytic virulence factor

    PubMed Central

    Steinberger, Jutta; Skern, Tim

    2016-01-01

    The leader proteinase (Lpro) of foot-and-mouth disease virus, inhibits the host innate immune response by at least three different mechanisms. The most well characterised is the prevention of the synthesis of cytokines such as interferons immediately after infection, brought about by specific proteolytic cleavage of the eukaryotic initiation factor 4G. This prevents the recruitment of capped cellular mRNA; the viral RNA can however be translated under these conditions. The two other mechanisms are the induction of NF-κB cleavage and the deubiquitination of immune signalling molecules. This review focuses on the structure-function relationships in Lpro responsible for these widely divergent activities. PMID:24670358

  20. A smallest 6 kda metalloprotease, mini-matrilysin, in living world: a revolutionary conserved zinc-dependent proteolytic domain- helix-loop-helix catalytic zinc binding domain (ZBD)

    PubMed Central

    2012-01-01

    Background The Aim of this study is to study the minimum zinc dependent metalloprotease catalytic folding motif, helix B Met loop-helix C, with proteolytic catalytic activities in metzincin super family. The metzincin super family share a catalytic domain consisting of a twisted five-stranded β sheet and three long α helices (A, B and C). The catalytic zinc is at the bottom of the cleft and is ligated by three His residues in the consensus sequence motif, HEXXHXXGXXH, which is located in helix B and part of the adjacent Met turn region. An interesting question is - what is the minimum portion of the enzyme that still possesses catalytic and inhibitor recognition?” Methods We have expressed a 60-residue truncated form of matrilysin which retains only the helix B-Met turn-helix C region and deletes helix A and the five-stranded β sheet which form the upper portion of the active cleft. This is only 1/4 of the full catalytic domain. The E. coli derived 6 kDa MMP-7 ZBD fragments were purified and refolded. The proteolytic activities were analyzed by Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay and CM-transferrin zymography analysis. SC44463, BB94 and Phosphoramidon were computationally docked into the 3day structure of the human MMP7 ZBD and TAD and thermolysin using the docking program GOLD. Results This minimal 6 kDa matrilysin has been refolded and shown to have proteolytic activity in the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay. Triton X-100 and heparin are important factors in the refolding environment for this mini-enzyme matrilysin. This minienzyme has the proteolytic activity towards peptide substrate, but the hexamer and octamer of the mini MMP-7 complex demonstrates the CM-transferrin proteolytic activities in zymographic analysis. Peptide digestion is inhibited by SC44463, specific MMP7 inhibitors, but not phosphorimadon. Interestingly, the mini MMP-7 can be processed by autolysis and producing ~ 6 ~ 7 kDa fragments. Thus

  1. The Eph Tyrosine Kinase Receptors EphB2 and EphA2 Are Novel Proteolytic Substrates of Tissue Factor/Coagulation Factor VIIa*

    PubMed Central

    Eriksson, Oskar; Ramström, Margareta; Hörnaeus, Katarina; Bergquist, Jonas; Mokhtari, Dariush; Siegbahn, Agneta

    2014-01-01

    Tissue factor (TF) binds the serine protease factor VIIa (FVIIa) to form a proteolytically active complex that can trigger coagulation or activate cell signaling. Here we addressed the involvement of tyrosine kinase receptors (RTKs) in TF/FVIIa signaling by antibody array analysis and subsequently found that EphB2 and EphA2 of the Eph RTK family were cleaved in their ectodomains by TF/FVIIa. We used N-terminal Edman sequencing and LC-MS/MS analysis to characterize the cleaved Eph isoforms and identified a key arginine residue at the cleavage site, in agreement with the tryptic serine protease activity of FVIIa. Protease-activated receptor 2 (PAR2) signaling and downstream coagulation activity was non-essential in this context, in further support of a direct cleavage by TF/FVIIa. EphB2 was cleaved by FVIIa concentrations in the subnanomolar range in a number of TF expressing cell types, indicating that the active cellular pool of TF was involved. FVIIa caused potentiation of cell repulsion by the EphB2 ligand ephrin-B1, demonstrating a novel proteolytical event to control Eph-mediated cell segregation. These results define Eph RTKs as novel proteolytical targets of TF/FVIIa and provide new insights into how TF/FVIIa regulates cellular functions independently of PAR2. PMID:25281742

  2. Cold Temperature Induces the Reprogramming of Proteolytic Pathways in Yeast.

    PubMed

    Isasa, Marta; Suñer, Clara; Díaz, Miguel; Puig-Sàrries, Pilar; Zuin, Alice; Bichman, Anne; Gygi, Steven P; Rebollo, Elena; Crosas, Bernat

    2016-01-22

    Despite much evidence of the involvement of the proteasome-ubiquitin signaling system in temperature stress response, the dynamics of the ubiquitylome during cold response has not yet been studied. Here, we have compared quantitative ubiquitylomes from a strain deficient in proteasome substrate recruitment and a reference strain during cold response. We have observed that a large group of proteins showing increased ubiquitylation in the proteasome mutant at low temperature is comprised by reverses suppressor of Ty-phenotype 5 (Rsp5)-regulated plasma membrane proteins. Analysis of internalization and degradation of plasma membrane proteins at low temperature showed that the proteasome becomes determinant for this process, whereas, at 30 °C, the proteasome is dispensable. Moreover, our observations indicate that proteasomes have increased capacity to interact with lysine 63-polyubiquitylated proteins during low temperature in vivo. These unanticipated observations indicate that, during cold response, there is a proteolytic cellular reprogramming in which the proteasome acquires a role in the endocytic-vacuolar pathway. PMID:26601941

  3. Addressing proteolytic efficiency in enzymatic degradation therapy for celiac disease

    PubMed Central

    Rey, Martial; Yang, Menglin; Lee, Linda; Zhang, Ye; Sheff, Joey G.; Sensen, Christoph W.; Mrazek, Hynek; Halada, Petr; Man, Petr; McCarville, Justin L; Verdu, Elena F.; Schriemer, David C.

    2016-01-01

    Celiac disease is triggered by partially digested gluten proteins. Enzyme therapies that complete protein digestion in vivo could support a gluten-free diet, but the barrier to completeness is high. Current options require enzyme amounts on the same order as the protein meal itself. In this study, we evaluated proteolytic components of the carnivorous pitcher plant (Nepenthes spp.) for use in this context. Remarkably low doses enhance gliadin solubilization rates, and degrade gliadin slurries within the pH and temporal constraints of human gastric digestion. Potencies in excess of 1200:1 (substrate-to-enzyme) are achieved. Digestion generates small peptides through nepenthesin and neprosin, the latter a novel enzyme defining a previously-unknown class of prolyl endoprotease. The digests also exhibit reduced TG2 conversion rates in the immunogenic regions of gliadin, providing a twin mechanism for evading T-cell recognition. When sensitized and dosed with enzyme-treated gliadin, NOD/DQ8 mice did not show intestinal inflammation, when compared to mice challenged with only pepsin-treated gliadin. The low enzyme load needed for effective digestion suggests that gluten detoxification can be achieved in a meal setting, using metered dosing based on meal size. We demonstrate this by showing efficient antigen processing at total substrate-to-enzyme ratios exceeding 12,000:1. PMID:27481162

  4. Proteolytic cleavage, trafficking, and functions of nuclear receptor tyrosine kinases.

    PubMed

    Chen, Mei-Kuang; Hung, Mien-Chie

    2015-10-01

    Intracellular localization has been reported for over three-quarters of receptor tyrosine kinase (RTK) families in response to environmental stimuli. Internalized RTK may bind to non-canonical substrates and affect various cellular processes. Many of the intracellular RTKs exist as fragmented forms that are generated by γ-secretase cleavage of the full-length receptor, shedding, alternative splicing, or alternative translation initiation. Soluble RTK fragments are stabilized and intracellularly transported into subcellular compartments, such as the nucleus, by binding to chaperone or transcription factors, while membrane-bound RTKs (full-length or truncated) are transported from the plasma membrane to the ER through the well-established Rab- or clathrin adaptor protein-coated vesicle retrograde trafficking pathways. Subsequent nuclear transport of membrane-bound RTK may occur via two pathways, INFS or INTERNET, with the former characterized by release of receptors from the ER into the cytosol and the latter characterized by release of membrane-bound receptor from the ER into the nucleoplasm through the inner nuclear membrane. Although most non-canonical intracellular RTK signaling is related to transcriptional regulation, there may be other functions that have yet to be discovered. In this review, we summarize the proteolytic processing, intracellular trafficking and nuclear functions of RTKs, and discuss how they promote cancer progression, and their clinical implications. PMID:26096795

  5. Stimulation of proteolytic digestion by intestinal goblet cell mucus.

    PubMed

    Shora, W; Forstner, G G; Forstner, J F

    1975-03-01

    Intestinal goblet cell mucus (GCM) was added to incubations of casein and trypsin (or chymotrypsin) to discover whether mucus could inhibit proteolysis. Contrary to expectation, GCM stimulated casein hydrolysis, reaching a maximum effect at a GCM to casein ratio (w/w) of 0.083. GCM did not contain proteolytic enzymes or proenzymes as contaminants, nor did GCM serve as a substrate for trypsin. Stimulation was not reduced by removing 85% of the sialic acid from GCM. Harsh physical treatment (boiling and freezing) of casein decreased (50%) the GCM effect, as did partial predigestion of casein by trypsin, and elevation of trypsin concentration beyond 3 mug per ml. Thus the undegraded structure of casein appeared to be important for the stimulation of proteolysis by GCM. GCM also enhanced the hydrolysis by trypsin of intestinal brush border membrane protein, but had no effect on the hydrolysis of hemoglobin, albumin, or benzoyl arginine ethyl ester. These results suggest that GCM reacts with specific substrates, in a fashion which promotes their digestion by trypsin or chymotrypsin. PMID:1112451

  6. Analysis of the proteolytic degradation products of hyaline cartilage proteoglycans.

    PubMed

    Liszt, F; Schnittker-Schulze, K; Stuhlsatz, H W; Greiling, H

    1990-01-01

    The proteolytic degradation products of nasal hyaline cartilage proteoglycans produced by polymorphonuclear leukocyte lysosomal enzymes were investigated. The protein content of the degradation products is 7.0-8.6% corresponding to a peptide chain of 24-28 amino acids and the relative molecular mass of the total fragment is M(r) = 37,600-39,200. On an average, each proteoglycan fragment contains two chondroitin-sulphate chains (M(r) = 22,000-22,400), every fourth fragment contains a keratan sulphate chain (M(r) = 7000-7200) and every seventh to eighth contains an O-glycosidic oligosaccharide. The results of the disaccharide analysis show that the galactosaminoglycan chains contain 76.2-83.6% chondroitin-4-sulphate, 12.9-19.4% chondroitin-6-sulphate, 3.5-3.8% chondroitin and no dermatan sulphate. Since composition and relative molecular mass of the chondroitin sulphate and keratan sulphate chains from the degradation products resemble those from native proteoglycans, it is suggested that the degradation of the proteoglycans occurs by proteinases that attack preferably the chondroitin sulphate region of the core protein. PMID:1726643

  7. Addressing proteolytic efficiency in enzymatic degradation therapy for celiac disease.

    PubMed

    Rey, Martial; Yang, Menglin; Lee, Linda; Zhang, Ye; Sheff, Joey G; Sensen, Christoph W; Mrazek, Hynek; Halada, Petr; Man, Petr; McCarville, Justin L; Verdu, Elena F; Schriemer, David C

    2016-01-01

    Celiac disease is triggered by partially digested gluten proteins. Enzyme therapies that complete protein digestion in vivo could support a gluten-free diet, but the barrier to completeness is high. Current options require enzyme amounts on the same order as the protein meal itself. In this study, we evaluated proteolytic components of the carnivorous pitcher plant (Nepenthes spp.) for use in this context. Remarkably low doses enhance gliadin solubilization rates, and degrade gliadin slurries within the pH and temporal constraints of human gastric digestion. Potencies in excess of 1200:1 (substrate-to-enzyme) are achieved. Digestion generates small peptides through nepenthesin and neprosin, the latter a novel enzyme defining a previously-unknown class of prolyl endoprotease. The digests also exhibit reduced TG2 conversion rates in the immunogenic regions of gliadin, providing a twin mechanism for evading T-cell recognition. When sensitized and dosed with enzyme-treated gliadin, NOD/DQ8 mice did not show intestinal inflammation, when compared to mice challenged with only pepsin-treated gliadin. The low enzyme load needed for effective digestion suggests that gluten detoxification can be achieved in a meal setting, using metered dosing based on meal size. We demonstrate this by showing efficient antigen processing at total substrate-to-enzyme ratios exceeding 12,000:1. PMID:27481162

  8. High Proteolytic Resistance of Spider-Derived Inhibitor Cystine Knots

    PubMed Central

    Kikuchi, Kyoko; Sugiura, Mika; Kimura, Tadashi

    2015-01-01

    Proteolytic stability in gastrointestinal tract and blood plasma is the major obstacle for oral peptide drug development. Inhibitor cystine knots (ICKs) are linear cystine knot peptides which have multifunctional properties and could become promising drug scaffolds. ProTx-I, ProTx-II, GTx1-15, and GsMTx-4 were spider-derived ICKs and incubated with pepsin, trypsin, chymotrypsin, and elastase in physiological conditions to find that all tested peptides were resistant to pepsin, and ProTx-II, GsMTx-4, and GTx1-15 showed resistance to all tested proteases. Also, no ProTx-II degradation was observed in rat blood plasma for 24 hours in vitro and ProTx-II concentration in circulation decreased to half in 40 min, indicating absolute stability in plasma and fast clearance from the system. So far, linear peptides are generally thought to be unsuitable in vivo, but all tested ICKs were not degraded by pepsin and stomach could be selected for the alternative site of drug absorption for fast onset of the drug action. Since spider ICKs are selective inhibitors of various ion channels which are related to the pathology of many diseases, engineered ICKs will make a novel class of peptide medicines which can treat variety of bothering symptoms. PMID:26843868

  9. Anti-proteolytic capacity and bonding durability of proanthocyanidin-biomodified demineralized dentin matrix

    PubMed Central

    Liu, Rui-Rui; Fang, Ming; Zhang, Ling; Tang, Cheng-Fang; Dou, Qi; Chen, Ji-Hua

    2014-01-01

    Our previous studies showed that biomodification of demineralized dentin collagen with proanthocyanidin (PA) for a clinically practical duration improves the mechanical properties of the dentin matrix and the immediate resin–dentin bond strength. The present study sought to evaluate the ability of PA biomodification to reduce collagenase-induced biodegradation of demineralized dentin matrix and dentin/adhesive interfaces in a clinically relevant manner. The effects of collagenolytic and gelatinolytic activity on PA-biomodified demineralized dentin matrix were analysed by hydroxyproline assay and gelatin zymography. Then, resin-/dentin-bonded specimens were prepared and challenged with bacterial collagenases. Dentin treated with 2% chlorhexidine and untreated dentin were used as a positive and negative control, respectively. Collagen biodegradation, the microtensile bond strengths of bonded specimens and the micromorphologies of the fractured interfaces were assessed. The results revealed that both collagenolytic and gelatinolytic activity on demineralized dentin were notably inhibited in the PA-biomodified groups, irrespective of PA concentration and biomodification duration. When challenged with exogenous collagenases, PA-biomodified bonded specimens exhibited significantly less biodegradation and maintained higher bond strengths than the untreated control. These results suggest that PA biomodification was effective at inhibiting proteolytic activity on demineralized dentin matrix and at stabilizing the adhesive/dentin interface against enzymatic degradation, is a new concept that has the potential to improve bonding durability. PMID:24810807

  10. Nrf2 silencing to inhibit proteolytic defense induced by hyperthermia in HT22 cells

    PubMed Central

    Bozaykut, Perinur; Ozer, Nesrin Kartal; Karademir, Betul

    2016-01-01

    Nrf2 pathway has been known to be protective against cancer progression however recent studies have revealed that the antioxidant activity of Nrf2 contributes to chemotherapy resistance. For many years, hyperthermia has been used as an additional therapy to increase the efficiency of chemotherapy and radiotherapy. Besides the positive effects of hyperthermia during treatment procedure, thermotolerance has been found to develop against heat treatment. Although the involved molecular mechanisms have not been fully clarified, heat shock proteins (HSP) and proteasome activity are known to be involved in the acquisition of thermotolerance. The aim of this study was to investigate the potential beneficial effects of combining hyperthermia with Nrf2 silencing to inhibit molecular mechanisms leading to induction of defense mechanisms in transcription level. Following heat treatment of HT22 cells, HSP70 and the proteasome levels and as well as proteasome activity were found to be elevated in the nucleus. Our results demonstrated that Nrf2 silencing reduced defense mechanisms against heat treatment both in antioxidant and proteolytic manner and Nrf2 may be a potential target for therapeutic approach in order to improve the beneficial effects of hyperthermia in cancer therapy. PMID:26966891

  11. Butyrate and bioactive proteolytic form of Wnt-5a regulate colonic epithelial proliferation and spatial development.

    PubMed

    Uchiyama, Kazuhiko; Sakiyama, Toshio; Hasebe, Takumu; Musch, Mark W; Miyoshi, Hiroyuki; Nakagawa, Yasushi; He, Tong-Chuan; Lichtenstein, Lev; Naito, Yuji; Itoh, Yoshito; Yoshikawa, Toshikazu; Jabri, Bana; Stappenbeck, Thaddeus; Chang, Eugene B

    2016-01-01

    Proliferation and spatial development of colonic epithelial cells are highly regulated along the crypt vertical axis, which, when perturbed, can result in aberrant growth and carcinogenesis. In this study, two key factors were identified that have important and counterbalancing roles regulating these processes: pericrypt myofibroblast-derived Wnt-5a and the microbial metabolite butyrate. Cultured YAMC cell proliferation and heat shock protein induction were analzyed after butryate, conditioned medium with Wnt5a activity, and FrzB containing conditioned medium. In vivo studies to modulate Hsp25 employed intra-colonic wall Hsp25 encoding lentivirus. To silence Wnt-5a in vivo, intra-colonic wall Wnt-5a silencing RNA was used. Wnt-5a, secreted by stromal myofibroblasts of the lower crypt, promotes proliferation through canonical β-catenin activation. Essential to this are two key requirements: (1) proteolytic conversion of the highly insoluble ~40 kD Wnt-5a protein to a soluble 36 mer amino acid peptide that activates epithelial β-catenin and cellular proliferation, and (2) the simultaneous inhibition of butyrate-induced Hsp25 by Wnt-5a which is necessary to arrest the proliferative process in the upper colonic crypt. The interplay and spatial gradients of these factors insures that crypt epithelial cell proliferation and development proceed in an orderly fashion, but with sufficient plasticity to adapt to physiological perturbations including inflammation. PMID:27561676

  12. Nrf2 silencing to inhibit proteolytic defense induced by hyperthermia in HT22 cells.

    PubMed

    Bozaykut, Perinur; Ozer, Nesrin Kartal; Karademir, Betul

    2016-08-01

    Nrf2 pathway has been known to be protective against cancer progression however recent studies have revealed that the antioxidant activity of Nrf2 contributes to chemotherapy resistance. For many years, hyperthermia has been used as an additional therapy to increase the efficiency of chemotherapy and radiotherapy. Besides the positive effects of hyperthermia during treatment procedure, thermotolerance has been found to develop against heat treatment. Although the involved molecular mechanisms have not been fully clarified, heat shock proteins (HSP) and proteasome activity are known to be involved in the acquisition of thermotolerance. The aim of this study was to investigate the potential beneficial effects of combining hyperthermia with Nrf2 silencing to inhibit molecular mechanisms leading to induction of defense mechanisms in transcription level. Following heat treatment of HT22 cells, HSP70 and the proteasome levels and as well as proteasome activity were found to be elevated in the nucleus. Our results demonstrated that Nrf2 silencing reduced defense mechanisms against heat treatment both in antioxidant and proteolytic manner and Nrf2 may be a potential target for therapeutic approach in order to improve the beneficial effects of hyperthermia in cancer therapy. PMID:26966891

  13. Butyrate and bioactive proteolytic form of Wnt-5a regulate colonic epithelial proliferation and spatial development

    PubMed Central

    Uchiyama, Kazuhiko; Sakiyama, Toshio; Hasebe, Takumu; Musch, Mark W.; Miyoshi, Hiroyuki; Nakagawa, Yasushi; He, Tong-Chuan; Lichtenstein, Lev; Naito, Yuji; Itoh, Yoshito; Yoshikawa, Toshikazu; Jabri, Bana; Stappenbeck, Thaddeus; Chang, Eugene B.

    2016-01-01

    Proliferation and spatial development of colonic epithelial cells are highly regulated along the crypt vertical axis, which, when perturbed, can result in aberrant growth and carcinogenesis. In this study, two key factors were identified that have important and counterbalancing roles regulating these processes: pericrypt myofibroblast-derived Wnt-5a and the microbial metabolite butyrate. Cultured YAMC cell proliferation and heat shock protein induction were analzyed after butryate, conditioned medium with Wnt5a activity, and FrzB containing conditioned medium. In vivo studies to modulate Hsp25 employed intra-colonic wall Hsp25 encoding lentivirus. To silence Wnt-5a in vivo, intra-colonic wall Wnt-5a silencing RNA was used. Wnt-5a, secreted by stromal myofibroblasts of the lower crypt, promotes proliferation through canonical β-catenin activation. Essential to this are two key requirements: (1) proteolytic conversion of the highly insoluble ~40 kD Wnt-5a protein to a soluble 36 mer amino acid peptide that activates epithelial β-catenin and cellular proliferation, and (2) the simultaneous inhibition of butyrate-induced Hsp25 by Wnt-5a which is necessary to arrest the proliferative process in the upper colonic crypt. The interplay and spatial gradients of these factors insures that crypt epithelial cell proliferation and development proceed in an orderly fashion, but with sufficient plasticity to adapt to physiological perturbations including inflammation. PMID:27561676

  14. Deterioration of white croaker (Pennahia argentata) meat thermally-induced gel products caused by proteolytic enzymes in the contaminated intestine and kidney.

    PubMed

    Ueki, Nobuhiko; Wan, Jianrong; Watabe, Shugo

    2016-05-15

    Thermally-induced gels were made from white croaker (Pennahia argentata) meat in the presence of its organ extracts by pre-heating at 40 and 65°C for 20 min and subsequent heating at 85°C for 20 min. The breaking strength of the gels decreased with increasing concentrations of the intestinal extracts accompanying decomposition of myosin heavy chains. However, no significant changes in the gel strength occurred when the kidney extract was added. The proteolytic activity in the intestinal extracts examined in the meat homogenate had a maximum at 60°C and pH 8.90. These results suggest that the intestinal rather than kidney proteolytic activities are responsible for gel softening known as a modori phenomenon. Thus, the removal of intestinal tracts is essential to maintain a high quality of surimi-based products. PMID:26775990

  15. In vitro potency determination of botulinum neurotoxin B based on its receptor-binding and proteolytic characteristics.

    PubMed

    Wild, Emina; Bonifas, Ursula; Klimek, Jolanta; Trösemeier, Jan-Hendrik; Krämer, Beate; Kegel, Birgit; Behrensdorf-Nicol, Heike A

    2016-08-01

    Botulinum neurotoxins (BoNTs) are the most potent toxins known. However, the paralytic effect caused by BoNT serotypes A and B is taken advantage of to treat different forms of dystonia and in cosmetic procedures. Due to the increasing areas of application, the demand for BoNTs A and B is rising steadily. Because of the high toxicity, it is mandatory to precisely determine the potency of every produced BoNT batch, which is usually accomplished by performing toxicity testing (LD50 test) in mice. Here we describe an alternative in vitro assay for the potency determination of the BoNT serotype B. In this assay, the toxin is first bound to its specific receptor molecules. After the proteolytic subunit of the toxin has been released and activated by chemical reduction, it is exposed to synaptobrevin, its substrate protein. Finally the proteolytic cleavage is quantified by an antibody-mediated detection of the neoepitope, reaching a detection limit below 0.1mouseLD50/ml. Thus, the assay, named BoNT/B binding and cleavage assay (BoNT/B BINACLE), takes into account the binding as well as the protease function of the toxin, thereby measuring its biological activity. PMID:27032463

  16. Proteolytic processing of Atg32 by the mitochondrial i-AAA protease Yme1 regulates mitophagy.

    PubMed

    Wang, Ke; Jin, Meiyan; Liu, Xu; Klionsky, Daniel J

    2013-11-01

    Mitophagy, the autophagic removal of mitochondria, occurs through a highly selective mechanism. In the yeast Saccharomyces cerevisiae, the mitochondrial outer membrane protein Atg32 confers selectivity for mitochondria sequestration as a cargo by the autophagic machinery through its interaction with Atg11, a scaffold protein for selective types of autophagy. The activity of mitophagy in vivo must be tightly regulated considering that mitochondria are essential organelles that produce most of the cellular energy, but also generate reactive oxygen species that can be harmful to cell physiology. We found that Atg32 was proteolytically processed at its C terminus upon mitophagy induction. Adding an epitope tag to the C terminus of Atg32 interfered with its processing and caused a mitophagy defect, suggesting the processing is required for efficient mitophagy. Furthermore, we determined that the mitochondrial i-AAA protease Yme1 mediated Atg32 processing and was required for mitophagy. Finally, we found that the interaction between Atg32 and Atg11 was significantly weakened in yme1∆ cells. We propose that the processing of Atg32 by Yme1 acts as an important regulatory mechanism of cellular mitophagy activity. PMID:24025448

  17. Internalization and proteolytic action of botulinum toxins in CNS neurons and astrocytes.

    PubMed

    Verderio, C; Coco, S; Rossetto, O; Montecucco, C; Matteoli, M

    1999-07-01

    Tetanus and botulinum toxins bind and are internalized at the neuromuscular junction. Botulinum neurotoxins (BoNTs) enter the cytosol at the motor nerve terminal; tetanus neurotoxin (TeNT) proceeds retroaxonally inside the motor axon to reach the spinal cord inhibitory interneurons. Although the major target of BoNTs is the peripheral cholinergic terminals, CNS neurons are susceptible to intoxication as well. We investigated the route of entry and the proteolytic activity of BoNT/B and BoNT/F in cultured hippocampal neurons and astrocytes. We show that, differently from TeNT, which enters hippocampal neurons via the process of synaptic vesicle (SV) recycling, BoNTs are internalized and cleave the substrate synaptobrevin/VAMP2 via a process independent of synaptic activity. Labeling of living neurons with Texas Red-conjugated BoNTs and fluoresceinated dextran revealed that these toxins enter hippocampal neurons via endocytic processes not mediated by SV recycling. Botulinum toxins also exploit endocytosis to enter cultured astrocytes, where they partially cleave cellubrevin, a ubiquitous synaptobrevin/VAMP isoform. These results indicate that, in spite of their closely related protein structure, TeNT and BoNTs use different routes to penetrate hippocampal neurons. These findings bear important implications for the identification of the protein receptors of clostridial toxins. PMID:10386990

  18. REGULATED PROTEOLYTIC PROCESSING OF TIE1 MODULATES LIGAND RESPONSIVENESS OF THE RECEPTOR TYROSINE KINASE TIE2

    PubMed Central

    Marron, Marie B; Singh, Harprit; Tahir, Tariq A; Kavumkal, Jais; Kim, Hak-Zoo; Koh, Gou Young; Brindle, Nicholas PJ

    2008-01-01

    Regulated ectodomain shedding followed by intramembrane proteolysis has recently been recognized as important in cell signaling and for degradation of several type I transmembrane proteins. The receptor tyrosine kinase Tie1 is known to undergo ectodomain cleavage generating a membrane tethered endodomain. Here we show Tie1 is a substrate for regulated intramembrane proteolysis. Following Tie1 ectodomain cleavage the newly formed 45 kDa endodomain undergoes additional proteolytic processing mediated by γ-secretase to generate an amino-terminally truncated 42 kDa fragment which is subsequently degraded by proteasomal activity. This sequential processing occurs constitutively and is stimulated by phorbol ester and vascular endothelial growth factor. To assess the biological significance of regulated Tie1 processing we analyzed its effects on angiopoietin signaling. Activation of ectodomain cleavage causes loss of phosphorylated Tie1 holoreceptor and generation of phosphorylated receptor fragments in the presence of COMP-Angiopoietin1. A key function of γ-secretase is in preventing accumulation of these phosphorylated fragments. We also find that regulated Tie1 processing modulates ligand responsiveness of the Tie-1-associated receptor Tie2. Activation of Tie1 ectodomain cleavage increases COMP-Angiopoietin1 activation of Tie2. This correlates with increased ability of Tie2 to bind ligand following shedding of the Tie1 extracellular domain. A similar enhancement of ligand activation of Tie2 is seen when Tie1 expression is suppressed by RNA interference. Together these data indicate that Tie1, via its extracellular domain, limits the ability of ligand to bind and activate Tie2. Furthermore the data suggests regulated processing of Tie1 may be an important mechanism for controlling signaling by Tie2. PMID:17728252

  19. Risk assessment of proteolytic Clostridium botulinum in canned foie gras.

    PubMed

    Membré, Jeanne-Marie; Diao, Moctar; Thorin, Chantal; Cordier, Grégoire; Zuber, François; André, Stéphane

    2015-10-01

    In this study, a risk assessment of proteolytic Clostridium botulinum in canned foie gras was performed, the number of illnesses per year in France due to C. botulinum in foie gras was estimated. Data on initial level in raw materials were collected at manufacturers and analysed using a Negative Binomial distribution. The effect of the usual foie gras heat treatment (equivalent time at 121 °C: F0=0.5 min) was considered at two levels: first, it led to an inactivation (estimated to 2.3 log); second it led to a spore injury and then to a spore inhibition. This latter effect was assessed by analysing data from a challenge test study carried out with Clostridium sporogenes spores in the foie gras product. The probability of spore recovering after thermal inhibition was estimated to 9.5×10(-8) (corresponding to 7.0 log). The data on the consumption pattern were collected on the French market. The Quantitative Microbiological Risk Assessment (QMRA) model and all the assumptions are reported in detail in the study. The initial contamination of raw materials, effect of thermal treatment on microbial inactivation and spore inhibition were handled mathematically using a probabilistic framework, considering only the variability dimension. The model was implemented in Excel and run through Monte Carlo simulation, using @Risk software. In parallel, epidemiological data collected from the French Institute for Public Health Surveillance during the period 2001-2012 were used to estimate an Appropriate Level Of Protection (ALOP) and then a Food Safety Objective (FSO): ALOP equalled to 2.5×10(-3) illnesses per million inhabitant per year, FSO equalled to 1.4×10(-9) foie gras portions containing C. botulinum spore (expressed in decimal logarithm, FSO=-8.9). The QMRA model output values were smaller, but on the same order of magnitude as these two figures: 8.0×10(-4) illnesses per million inhabitants per year, and, 4.5×10(-10) (-9.3 log) foie gras portions containing C

  20. Kallikrein-related Peptidase 5 Functions in Proteolytic Processing of Profilaggrin in Cultured Human Keratinocytes*

    PubMed Central

    Sakabe, Jun-ichi; Yamamoto, Mami; Hirakawa, Satoshi; Motoyama, Akira; Ohta, Isao; Tatsuno, Kazuki; Ito, Taisuke; Kabashima, Kenji; Hibino, Toshihiko; Tokura, Yoshiki

    2013-01-01

    Filaggrin protein is synthesized in the stratum granulosum of the skin and contributes to the formation of the human skin barrier. Profilaggrin is cleaved by proteolytic enzymes and converted to functional filaggrin, but its processing mechanism remains not fully elucidated. Kallikrein-related peptidase 5 (KLK5) is a major serine protease found in the skin, which is secreted from lamellar granules following its expression in the stratum granulosum and activated in the extracellular space of the stratum corneum. Here, we searched for profilaggrin-processing protease(s) by partial purification of epidermal extracts and found KLK5 as a possible candidate. We used high performance liquid chromatography coupled with electrospray tandem mass spectrometry to show that KLK5 cleaves profilaggrin. Furthermore, based on a proximity ligation assay, immunohistochemistry, and immunoelectron microscopy analysis, we reveal that KLK5 and profilaggrin co-localize in the stratum granulosum in human epidermis. KLK5 knockdown in normal cultured human epidermal keratinocytes resulted in higher levels of profilaggrin, indicating that KLK5 potentially functions in profilaggrin cleavage. PMID:23629652

  1. A Peptide-Based Mechano-sensitive, Proteolytically Stable Hydrogel with Remarkable Antibacterial Properties.

    PubMed

    Baral, Abhishek; Roy, Subhasish; Ghosh, Srabanti; Hermida-Merino, Daniel; Hamley, Ian W; Banerjee, Arindam

    2016-02-23

    A long-chain amino acid containing dipeptide has been found to form a hydrogel in phosphate buffer whose pH ranges from 6.0 to 8.8. The hydrogel formed at pH 7.46 has been characterized by small-angle X-ray scattering (SAXS), wide-angle powder X-ray diffraction (PXRD), Fourier transform infrared (FT-IR) spectroscopy, field-emission scanning electron microscopy (FE-SEM), high-resolution transmission electron microscopy (HR-TEM) imaging and rheological analyses. The microscopic imaging studies suggest the formation of a nanofibrillar three-dimensional (3D) network for the hydrogel. As observed visually and confirmed rheologically, the hydrogel at pH 7.46 exhibits thixotropy. This thixotropic property can be exploited to inject the peptide. Furthermore, the hydrogel exhibits remarkable antibacterial activity against Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, which are responsible for many common diseases. The hydrogel has practical applicability due to its biocompatibility with human red blood cells and human fibroblast cells. Interestingly, this hydrogel shows high resistance toward proteolytic enzymes, making it a new potential antimicrobial agent for future applications. It has also been observed that a small change in molecular structure of the gelator peptide not only turns the gelator into a nongelator molecule under similar conditions, but it also has a significant negative impact on its bactericidal character. PMID:26818698

  2. Hypoxia Associated Proteolytic Processing of OS-9 by the Metalloproteinase Meprin β

    PubMed Central

    Martin, Barry Lee; Conley, Sabena Michelle; Harris, Regine Simone; Stanley, Corshe Devon

    2016-01-01

    Meprin metalloproteases play a role in the pathology of ischemia/reperfusion- (IR-) induced renal injury. The endoplasmic reticulum-associated protein, osteosarcoma-9 (OS-9), has been shown to interact with the carboxyl-terminal tail of meprin β. More importantly, OS-9 interacts with the hypoxia inducible factor-1α (HIF-1α) and the prolyl-hydroxylase, proteins which mediate the cell's response to hypoxia. To determine if OS-9 is a meprin substrate, kidney proteins from meprin αβ knockout mice (αβKO) (which lack endogenous meprins) and purified human OS-9 were incubated with activated forms of meprin A and meprin B, and Western blot analysis was used to evaluate proteolytic processing of OS-9. Fragmentation of OS-9 was observed in reactions with meprin B, but not meprin A. To determine whether meprin B cleaves OS-9 in vivo, wild-type (WT) and meprin αβKO mice were subjected to IR-induced renal injury. Fragmentation of OS-9 was observed in kidney proteins from WT mice subjected to IR, but not in meprin αβKO counterparts. Transfection of kidney cells (MDCK and HEK293) with meprin β cDNA prevented accumulation of OS-9 following exposure to the hypoxia mimic, CoCl2. These data suggest that meprin β interaction with OS-9 plays a role in the hypoxia response associated with IR-induced renal injury. PMID:27478637

  3. Hypoxia Associated Proteolytic Processing of OS-9 by the Metalloproteinase Meprin β.

    PubMed

    Martin, Barry Lee; Conley, Sabena Michelle; Harris, Regine Simone; Stanley, Corshe Devon; Niyitegeka, Jean-Marie Vianney; Ongeri, Elimelda Moige

    2016-01-01

    Meprin metalloproteases play a role in the pathology of ischemia/reperfusion- (IR-) induced renal injury. The endoplasmic reticulum-associated protein, osteosarcoma-9 (OS-9), has been shown to interact with the carboxyl-terminal tail of meprin β. More importantly, OS-9 interacts with the hypoxia inducible factor-1α (HIF-1α) and the prolyl-hydroxylase, proteins which mediate the cell's response to hypoxia. To determine if OS-9 is a meprin substrate, kidney proteins from meprin αβ knockout mice (αβKO) (which lack endogenous meprins) and purified human OS-9 were incubated with activated forms of meprin A and meprin B, and Western blot analysis was used to evaluate proteolytic processing of OS-9. Fragmentation of OS-9 was observed in reactions with meprin B, but not meprin A. To determine whether meprin B cleaves OS-9 in vivo, wild-type (WT) and meprin αβKO mice were subjected to IR-induced renal injury. Fragmentation of OS-9 was observed in kidney proteins from WT mice subjected to IR, but not in meprin αβKO counterparts. Transfection of kidney cells (MDCK and HEK293) with meprin β cDNA prevented accumulation of OS-9 following exposure to the hypoxia mimic, CoCl2. These data suggest that meprin β interaction with OS-9 plays a role in the hypoxia response associated with IR-induced renal injury. PMID:27478637

  4. Mitochondrial proteolytic stress induced by loss of mortalin function is rescued by Parkin and PINK1

    PubMed Central

    Burbulla, L F; Fitzgerald, J C; Stegen, K; Westermeier, J; Thost, A-K; Kato, H; Mokranjac, D; Sauerwald, J; Martins, L M; Woitalla, D; Rapaport, D; Riess, O; Proikas-Cezanne, T; Rasse, T M; Krüger, R

    2014-01-01

    The mitochondrial chaperone mortalin was implicated in Parkinson's disease (PD) because of its reduced levels in the brains of PD patients and disease-associated rare genetic variants that failed to rescue impaired mitochondrial integrity in cellular knockdown models. To uncover the molecular mechanisms underlying mortalin-related neurodegeneration, we dissected the cellular surveillance mechanisms related to mitochondrial quality control, defined the effects of reduced mortalin function at the molecular and cellular levels and investigated the functional interaction of mortalin with Parkin and PINK1, two PD-related proteins involved in mitochondrial homeostasis. We found that reduced mortalin function leads to: (1) activation of the mitochondrial unfolded protein response (UPR(mt)), (2) increased susceptibility towards intramitochondrial proteolytic stress, (3) increased autophagic degradation of fragmented mitochondria and (4) reduced mitochondrial mass in human cells in vitro and ex vivo. These alterations caused increased vulnerability toward apoptotic cell death. Proteotoxic perturbations induced by either partial loss of mortalin or chemical induction were rescued by complementation with native mortalin, but not disease-associated mortalin variants, and were independent of the integrity of autophagic pathways. However, Parkin and PINK1 rescued loss of mortalin phenotypes via increased lysosomal-mediated mitochondrial clearance and required intact autophagic machinery. Our results on loss of mortalin function reveal a direct link between impaired mitochondrial proteostasis, UPR(mt) and PD and show that effective removal of dysfunctional mitochondria via either genetic (PINK1 and Parkin overexpression) or pharmacological intervention (rapamycin) may compensate mitochondrial phenotypes. PMID:24743735

  5. Proteolytic Cleavage of the Plasmodium falciparum Circumsporozoite Protein Is a Target of Protective Antibodies.

    PubMed

    Espinosa, Diego A; Gutierrez, Gabriel M; Rojas-López, Maricarmen; Noe, Amy R; Shi, Lirong; Tse, Sze-Wah; Sinnis, Photini; Zavala, Fidel

    2015-10-01

    Studies in animals and human volunteers demonstrate that antibodies against the repeat-region of the Plasmodium circumsporozoite protein (CSP) abrogate sporozoite infection. However, the realization that the N- and C- terminal regions flanking the repeats play essential roles in parasite infectivity raised the possibility that they could be targeted by protective antibodies. We characterized a monoclonal antibody (mAb5D5) specific for the N-terminus of the P. falciparum CSP, which inhibits the proteolytic cleavage of the CSP, a key requirement for parasite infection of hepatocytes. Adoptive transfer of mAb5D5 strongly inhibits the in vivo infection of sporozoites expressing the N-terminus of P. falciparum CSP, and this protection is greatly enhanced when combined with antirepeat antibodies. Our results show that antibodies interfering with molecular processes required for parasite infectivity can exert a strong in vivo protective activity and indicate that pre-erythrocytic vaccines against Plasmodium should include the CSP N-terminal region. PMID:25762791

  6. Leucoagaricus gongylophorus uses leaf-cutting ants to vector proteolytic enzymes towards new plant substrate

    PubMed Central

    Kooij, Pepijn W; Rogowska-Wrzesinska, Adelina; Hoffmann, Daniel; Roepstorff, Peter; Boomsma, Jacobus J; Schiøtt, Morten

    2014-01-01

    The mutualism between leaf-cutting ants and their fungal symbionts revolves around processing and inoculation of fresh leaf pulp in underground fungus gardens, mediated by ant fecal fluid deposited on the newly added plant substrate. As herbivorous feeding often implies that growth is nitrogen limited, we cloned and sequenced six fungal proteases found in the fecal fluid of the leaf-cutting ant Acromyrmex echinatior and identified them as two metalloendoproteases, two serine proteases and two aspartic proteases. The metalloendoproteases and serine proteases showed significant activity in fecal fluid at pH values of 5–7, but the aspartic proteases were inactive across a pH range of 3–10. Protease activity disappeared when the ants were kept on a sugar water diet without fungus. Relative to normal mycelium, both metalloendoproteases, both serine proteases and one aspartic protease were upregulated in the gongylidia, specialized hyphal tips whose only known function is to provide food to the ants. These combined results indicate that the enzymes are derived from the ingested fungal tissues. We infer that the five proteases are likely to accelerate protein extraction from plant cells in the leaf pulp that the ants add to the fungus garden, but regulatory functions such as activation of proenzymes are also possible, particularly for the aspartic proteases that were present but without showing activity. The proteases had high sequence similarities to proteolytic enzymes of phytopathogenic fungi, consistent with previous indications of convergent evolution of decomposition enzymes in attine ant fungal symbionts and phytopathogenic fungi. PMID:24401858

  7. Host Defense Functions of Proteolytically Processed and Parent (Unprocessed) Cathelicidins of Rabbit Granulocytes

    PubMed Central

    Zarember, Kol A.; Katz, Seth S.; Tack, Brian F.; Doukhan, Laurence; Weiss, Jerrold; Elsbach, Peter

    2002-01-01

    Members of the cathelicidin family are present in all mammals studied. Generally, these proteins contain a conserved N-terminal domain and a structurally and functionally divergent C-terminal region that expresses antibacterial or other activities when proteolytically released. Rabbit granulocytes produce CAP18, a cathelicidin that conforms to this structural and functional organization, and also 15-kDa protein isoforms (p15s) that share several key structural features with other cathelicidins but apparently do not undergo processing with release of an active peptide. To further define the importance of proteolysis in the antibacterial activities of these proteins, we have purified from granulocytes proCAP18, its C-terminal peptide (CAP18p), and two p15 isoforms to apparent homogeneity. Of these four polypeptides, only CAP18p was independently cytotoxic to encapsulated Escherichia coli (90% inhibitory concentration, ∼600 nM) but it was ∼50-fold less potent on a molar basis than the bactericidal/permeability-increasing protein (BPI). However, all four cathelicidin species, notably including proCAP18, exhibited antibacterial synergy with BPI, and the p15s also displayed synergy with CAP18p in the absence of BPI. Subnanomolar concentrations of proCAP18 blocked lipopolysaccharide-induced chemiluminescence of human leukocytes, showing a molar potency more than 100-fold greater than that of CAP18p (∼20 nM) or BPI (∼50 nM). Thus, while independent bactericidal activity of cathelicidins requires processing, other host-defense functions do not and are more potently expressed by the unprocessed protein than by the C-terminal peptide. PMID:11796584

  8. Effect on platelet FXIII and partial characterization of Lonomin V, a proteolytic enzyme from Lonomia achelous caterpillars.

    PubMed

    Guerrero, B; Perales, J; Gil, A; Arocha-Piñango, C L

    1999-03-01

    Contact with Lonomia achelous caterpillars venom induces a severe bleeding syndrome in humans. A constant finding in all reported cases is a marked decrease of blood coagulation factor XIII (FXIII), which has been attributed to the presence of a proteolytic enzyme, isolated and named Lonomin V, in the hemolymph and hair secretion. In this study, the effect of Lonomin V on transglutaminase activity from human plasma, rabbit plasma, and platelet FXIII was analyzed. The decrease of activity was more pronounced in platelet (A2) when compared with rabbit plasma (AB) and human plasma FXIII (A2B2). This finding might be explained by the differences in FXIII molecular structure. In addition, platelet FXIII molecule was degraded by Lonomin to several fragments of low molecular mass. Lonomin V was stable over a wide range of pH (6-8.5) and temperatures of -70 degrees C, -20 degrees C and between 4 to 24 degrees C, with a progressive decrease at 37 degrees C and total inactivation at 60 degrees C after 2 hours incubation. Diisopropyl fluoro-phosphate, phenylmethylsulfonyl fluoride, tosyl-l-lysine chloromethyl ketone, and iodoacetamide abolished the effect of Lonomin V on FXIII; in contrast dithiothreitol and EDTA-Na enhance the activity. We concluded that Lonomin V is a serine proteinase with a free Cys essential for the enzymatic activity. Due to its proteolytic activity on FXIII, with concomitant impairment of fibrin cross-linking, Lonomin V might be useful in association with thrombolytic drugs for preventing rethrombosis. PMID:10074908

  9. Improvement of proteolytic and oxidative stability of Chondroitinase ABC I by cosolvents.

    PubMed

    Nazari-Robati, Mahdieh; Golestani, Abolfazl; Asadikaram, GholamReza

    2016-10-01

    Recently, utilization of the enzyme Chondroitinase ABC I (cABC I) has received considerable attention in treatment of spinal cord injury. cABC I removes chondroitin sulfate proteoglycans which are inhibitory to axon growth and enhances nerve regeneration. Therefore, determination of cABC I resistance to proteolysis and oxidation provides valuable information for optimizing its clinical application. In this work, proteolytic stability of cABC I to trypsin and chymotrypsin as well as its oxidative resistance to H2O2 was measured. Moreover, the effect of cosolvents glycerol, sorbitol and trehalose on cABC I proteolytic and oxidative stability was determined. The results indicated that cABC I is highly susceptible to proteolysis and oxidation. Comparison of proteolytic patterns demonstrated a high degree of similarity which confirmed the exposure of specific regions of cABC I to proteolysis. However, proteolytic degradation was significantly reduced in the presence of cosolvents. In addition, cosolvents decreased the rate of both cABC I proteolytic and oxidative inactivation. Notably, the degree of stabilization provided by these cosolvents varied greatly. These findings indicated the high potential of cosolvents in protein stabilization to proteolysis and oxidative inactivation. PMID:27311501

  10. Dynamics of digestive proteolytic system during blood feeding of the hard tick Ixodes ricinus

    PubMed Central

    2010-01-01

    Background Ticks are vectors of a wide variety of pathogens causing severe diseases in humans and domestic animals. Intestinal digestion of the host blood is an essential process of tick physiology and also a limiting factor for pathogen transmission since the tick gut represents the primary site for pathogen infection and proliferation. Using the model tick Ixodes ricinus, the European Lyme disease vector, we have previously demonstrated by genetic and biochemical analyses that host blood is degraded in the tick gut by a network of acidic peptidases of the aspartic and cysteine classes. Results This study reveals the digestive machinery of the I. ricinus during the course of blood-feeding on the host. The dynamic profiling of concentrations, activities and mRNA expressions of the major digestive enzymes demonstrates that the de novo synthesis of peptidases triggers the dramatic increase of the hemoglobinolytic activity along the feeding period. Overall hemoglobinolysis, as well as the activity of digestive peptidases are negligible at the early stage of feeding, but increase dramatically towards the end of the slow feeding period, reaching maxima in fully fed ticks. This finding contradicts the established opinion that blood digestion is reduced at the end of engorgement. Furthermore, we show that the digestive proteolysis is localized intracellularly throughout the whole duration of feeding. Conclusions Results suggest that the egressing proteolytic system in the early stage of feeding and digestion is a potential target for efficient impairment, most likely by blocking its components via antibodies present in the host blood. Therefore, digestive enzymes are promising candidates for development of novel 'anti-tick' vaccines capable of tick control and even transmission of tick-borne pathogens. PMID:21156061

  11. Lipopolysaccharide Induces Degradation of Connexin43 in Rat Astrocytes via the Ubiquitin-Proteasome Proteolytic Pathway

    PubMed Central

    Liao, Chih-Kai; Jeng, Chung-Jiuan; Wang, Hwai-Shi; Wang, Shu-Huei; Wu, Jiahn-Chun

    2013-01-01

    The astrocytic syncytium plays a critical role in maintaining the homeostasis of the brain through the regulation of gap junction intercellular communication (GJIC). Changes to GJIC in response to inflammatory stimuli in astrocytes may have serious effects on the brain. We have previously shown that lipopolysaccharide (LPS) reduces connexin43 (Cx43) expression and GJIC in cultured rat astrocytes via a toll-like receptor 4-mediated signaling pathway. In the present study, treatment of astrocytes with LPS resulted in a significant increase in levels of the phosphorylated forms of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) -1, -2, and -3 for up to 18 h. An increase in nuclear transcription factor NF-κB levels was also observed after 8 h of LPS treatment and was sustained for up to 18 h. The LPS-induced decrease in Cx43 protein levels and inhibition of GJIC were blocked by the SAPK/JNK inhibitor SP600125, but not by the NF-κB inhibitor BAY11-7082. Following blockade of de novo protein synthesis by cycloheximide, LPS accelerated Cx43 degradation. Moreover, the LPS-induced downregulation of Cx43 was blocked following inhibition of 26S proteasome activity using the reversible proteasome inhibitor MG132 or the irreversible proteasome inhibitor lactacystin. Immunoprecipitation analyses revealed an increased association of Cx43 with both ubiquitin and E3 ubiquitin ligase Nedd4 in astrocytes after LPS stimulation for 6 h and this effect was prevented by SP600125. Taken together, these results suggest that LPS stimulation leads to downregulation of Cx43 expression and GJIC in rat astrocytes by activation of SAPK/JNK and the ubiquitin-proteasome proteolytic pathway. PMID:24236122

  12. [Proteolytic enzymes from Streptomyces fradiae: a metalloendopeptidase, subtilisin-like, and trypsin-like proteinases].

    PubMed

    Bormatova, M E; Ivanova, N M; Iusupova, M P; Voiushina, T L; Surova, I A; Chestukhina, G G; Stepanov, V M

    1996-02-01

    Three proteolytic enzymes-the metalloproteinase, SFMP, and two serine proteinases, SFSP and SFTP-have been isolated and purified from the culture fluid of Streptomyces fradiae using chromatography on bacitracin-silochrome, bacitracin-Sepharose, DEAE-cellulose and fractionation by ammonium sulfate. Study of physico-chemical and functional properties of the enzymes and structural analysis revealed that SFMP is a cysteine-containing metalloendopeptidase with M(r) of 36 kDa, has a peak activity for synthetic substrates at pH 7.0-7.5 and at 60-65 degrees C and is stable at pH 7.0-9.0. The serine proteinase SFSP is related to subtilisin-like enzymes, has a M(r) of 29 kDa and a pH optimum at 7.5-8.5 at temperature up to 50 degrees C. The proteinase is stable at pH 4.0-9.0 and retains 30% of its activity at 70 degrees C. The other serine proteinase, SFTP, has a M(r) of 26 kDa and is related to trypsin-like enzymes. Its activity for synthetic substrates of trypsin is maximal at pH 6.8-8.8 at 50 degrees C. The enzyme is stable at pH 4.5-8.5 and at temperature below 50 degrees C. It has been shown that Streptomyces fradiae, like Streptomyces griseus and other Streptomycetes, possesses an ability to secrete serine proteinases (SFSP and SFTP) related to two evolutionally distinct families of serine proteinases, i.e., subtilisin and chymotrypsin families. SFMP and SFSP have been isolated and characterized for the first time. PMID:8717499

  13. Fangchinoline Inhibits Human Immunodeficiency Virus Type 1 Replication by Interfering with gp160 Proteolytic Processing

    PubMed Central

    Wan, Zhitao; Lu, Yimei; Liao, Qingjiao; Wu, Yang; Chen, Xulin

    2012-01-01

    The introduction of highly active antiretroviral therapy has led to a significant reduction in the morbidity and mortality of acquired immunodeficiency syndrome patients. However, the emergence of drug resistance has resulted in the failure of treatments in large numbers of patients and thus necessitates the development of new classes of anti-HIV drugs. In this study, more than 200 plant-derived small-molecule compounds were evaluated in a cell-based HIV-1 antiviral screen, resulting in the identification of a novel HIV-1 inhibitor (fangchinoline). Fangchinoline, a bisbenzylisoquinoline alkaloid isolated from Radix Stephaniae tetrandrae, exhibited antiviral activity against HIV-1 laboratory strains NL4-3, LAI and BaL in MT-4 and PM1 cells with a 50% effective concentration ranging from 0.8 to 1.7 µM. Mechanism-of-action studies showed that fangchinoline did not exhibit measurable antiviral activity in TZM-b1 cells but did inhibit the production of infectious virions in HIV-1 cDNA transfected 293T cells, which suggests that the compound targets a late event in infection cycle. Furthermore, the antiviral effect of fangchinoline seems to be HIV-1 enve1ope-dependent, as the production of infectious HIV-1 particles packaged with a heterologous envelope, the vesicular stomatitis virus G glycoprotein, was unaffected by fangchinoline. Western blot analysis of HIV envelope proteins expressed in transfected 293T cells and in isolated virions showed that fangchinoline inhibited HIV-1 gp160 processing, resulting in reduced envelope glycoprotein incorporation into nascent virions. Collectively, our results demonstrate that fangchinoline inhibits HIV-1 replication by interfering with gp160 proteolytic processing. Fangchinoline may serve as a starting point for developing a new HIV-1 therapeutic approach. PMID:22720080

  14. Fangchinoline inhibits human immunodeficiency virus type 1 replication by interfering with gp160 proteolytic processing.

    PubMed

    Wan, Zhitao; Lu, Yimei; Liao, Qingjiao; Wu, Yang; Chen, Xulin

    2012-01-01

    The introduction of highly active antiretroviral therapy has led to a significant reduction in the morbidity and mortality of acquired immunodeficiency syndrome patients. However, the emergence of drug resistance has resulted in the failure of treatments in large numbers of patients and thus necessitates the development of new classes of anti-HIV drugs. In this study, more than 200 plant-derived small-molecule compounds were evaluated in a cell-based HIV-1 antiviral screen, resulting in the identification of a novel HIV-1 inhibitor (fangchinoline). Fangchinoline, a bisbenzylisoquinoline alkaloid isolated from Radix Stephaniae tetrandrae, exhibited antiviral activity against HIV-1 laboratory strains NL4-3, LAI and BaL in MT-4 and PM1 cells with a 50% effective concentration ranging from 0.8 to 1.7 µM. Mechanism-of-action studies showed that fangchinoline did not exhibit measurable antiviral activity in TZM-b1 cells but did inhibit the production of infectious virions in HIV-1 cDNA transfected 293T cells, which suggests that the compound targets a late event in infection cycle. Furthermore, the antiviral effect of fangchinoline seems to be HIV-1 envelope-dependent, as the production of infectious HIV-1 particles packaged with a heterologous envelope, the vesicular stomatitis virus G glycoprotein, was unaffected by fangchinoline. Western blot analysis of HIV envelope proteins expressed in transfected 293T cells and in isolated virions showed that fangchinoline inhibited HIV-1 gp160 processing, resulting in reduced envelope glycoprotein incorporation into nascent virions. Collectively, our results demonstrate that fangchinoline inhibits HIV-1 replication by interfering with gp160 proteolytic processing. Fangchinoline may serve as a starting point for developing a new HIV-1 therapeutic approach. PMID:22720080

  15. Broad coverage identification of multiple proteolytic cleavage site sequences in complex high molecular weight proteins using quantitative proteomics as a complement to edman sequencing.

    PubMed

    Doucet, Alain; Overall, Christopher M

    2011-05-01

    Proteolytic processing modifies the pleiotropic functions of many large, complex, and modular proteins and can generate cleavage products with new biological activity. The identification of exact proteolytic cleavage sites in the extracellular matrix laminins, fibronectin, and other extracellular matrix proteins is not only important for understanding protein turnover but is needed for the identification of new bioactive cleavage products. Several such products have recently been recognized that are suggested to play important cellular regulatory roles in processes, including angiogenesis. However, identifying multiple cleavage sites in extracellular matrix proteins and other large proteins is challenging as N-terminal Edman sequencing of multiple and often closely spaced cleavage fragments on SDS-PAGE gels is difficult, thus limiting throughput and coverage. We developed a new liquid chromatography-mass spectrometry approach we call amino-terminal oriented mass spectrometry of substrates (ATOMS) for the N-terminal identification of protein cleavage fragments in solution. ATOMS utilizes efficient and low cost dimethylation isotopic labeling of original N-terminal and proteolytically generated N termini of protein cleavage fragments followed by quantitative tandem mass spectrometry analysis. Being a peptide-centric approach, ATOMS is not dependent on the SDS-PAGE resolution limits for protein fragments of similar mass. We demonstrate that ATOMS reliably identifies multiple proteolytic sites per reaction in complex proteins. Fifty-five neutrophil elastase cleavage sites were identified in laminin-1 and fibronectin-1 with 34 more identified by matrix metalloproteinase cleavage. Hence, our degradomics approach offers a complimentary alternative to Edman sequencing with broad applicability in identifying N termini such as cleavage sites in complex high molecular weight extracellular matrix proteins after in vitro cleavage assays. ATOMS can therefore be useful in

  16. Resin-assisted Enrichment of N-terminal Peptides for Characterizing Proteolytic Processing

    SciTech Connect

    Kim, Jong Seo; Dai, Ziyu; Aryal, Uma K.; Moore, Ronald J.; Camp, David G.; Baker, Scott E.; Smith, Richard D.; Qian, Weijun

    2013-06-17

    Proteolytic processing is a ubiquitous, irreversible posttranslational modification that plays an important role in cellular regulation in all living organisms. Herein we report a resin-assisted positive selection method for specifically enriching protein N-terminal peptides to facilitate the characterization of proteolytic processing events by liquid chromatography-tandem mass spectrometry. In this approach, proteins are initially reduced and alkylated and their lysine residues are converted to homoarginines. Then, protein N-termini are selectively converted to reactive thiol groups. We demonstrate that these sequential reactions were achieved with nearly quantitative efficiencies. Thiol-containing N-terminal peptides are then captured (>98% efficiency) by a thiol-affinity resin, a significant improvement over the traditional avidin/biotin enrichment. Application to cell lysates of Aspergillus niger, a filamentous fungus of interest for biomass degradation, enabled the identification of 1672 unique protein N-termini and proteolytic cleavage sites from 690 unique proteins.

  17. Proteolytic Equilibria of Vanillic Acid in the Ground and Excited States

    NASA Astrophysics Data System (ADS)

    Vusovich, O. V.; Tchaikovskaya, O. N.; Sokolova, I. V.; Vasil‧eva, N. Yu.

    2016-03-01

    Proteolytic equilibria of vanillic acid in aqueous solutions were studied using electronic spectroscopy. The pH ranges for anionic, dianionic, cationic, and neutral forms of vanillic acid in the ground and excited states were determined. The electron density distribution on atoms in the proteolytic forms was determined using quantum-chemistry methods. The anion formed as a result of dissociation of the carboxylic acid. The dianion formed in the presence of two and more equivalents of alkali as a result of proton loss from the phenol and carboxylic acid. The vanillic acid cation formed via protonation of the carbonyl oxygen. Differences in spectral features of the proteolytic forms in the ground and excited states were observed.

  18. Facile electrochemical detection of botulinum neurotoxin type E using a two-step proteolytic cleavage.

    PubMed

    Park, Seonhwa; Shin, Yu Mi; Song, Ji-Joon; Yang, Haesik

    2015-10-15

    Facile electrochemical methods for measuring protease concentration or protease activity are essential for point-of-care testing of toxic proteases. However, electrochemical detection of proteases, such as botulinum neurotoxin type E (BoNT/E), that cleave a peptide bond between two specific amino acid residues is challenging. This study reports a facile and sensitive electrochemical method for BoNT/E detection. The method is based on a two-step proteolytic cleavage using a target BoNT/E light chain (BoNT/E-LC) and an externally supplemented exopeptidase, L-leucine-aminopeptidase (LAP). BoNT/E-LC cleaves a peptide bond between arginine and isoleucine in IDTQNRQIDRI-4-amino-1-naphthol (oligopeptide-AN) to generate isoleucine-AN. Subsequently, LAP cleaves a bond between isoleucine and AN to liberate a free electroactive AN species. The liberated AN participates in electrochemical-chemical-chemical (ECC) redox cycling involving Ru(NH3)6(3+), AN, and a reducing agent, which allows a high signal amplification. Electrochemical detection is carried out without surface modification of indium-tin oxide electrodes. We show that dithiothreitol is beneficial for enhancing the enzymatic activity of BoNT/E-LC and also for achieving a fast ECC redox cycling. An incubation temperature of 37°C and the use of phosphate buffered saline (PBS) buffer resulted in optimal signal-to-background ratios for efficient BoNT/E detection. BoNT/E-LC could be detected at concentrations of approximately 2.0 pg/mL, 0.2, and 3 ng/mL after 4h, 2h, and 15 min incubation in PBS buffer, respectively, and approximately 0.3 ng/mL after 2-h incubation in bottled water. The method developed could be applied in fast, sensitive, and selective detection of any protease that cleaves a peptide bond between two specific amino acid residues. PMID:25982730

  19. The role of extracellular and intracellular proteolytic systems in aneurysms of the ascending aorta.

    PubMed

    Werner, Isabella; Schack, Stephanie; Richter, Manfred; Stock, Ulrich A; Ahmad, Ali El-Sayed; Moritz, Anton; Beiras-Fernandez, Andres

    2016-05-01

    Aneurysms of the ascending aorta are an outstanding challenge to clinicians as they may persist asymptomatic until they present with dissection or rupture. Intensive research is performed to reveal the molecular mechanisms causing aneurysm formation. Calpains are ubiquitous non-lysosomal cysteine proteases which are classically activated by calcium signaling. The two major forms of the calpain-family are calpain-I and calpain-II. Calpastatin specifically inhibits the proteolytic activity of calpain-I and -II. Recently it has been demonstrated in aneurysm tissues from ascending aortas obtained from Marfan syndrome patients that calpain-II expression is increased and calpastatin expression is decreased. Thus, we were interested in the probable role of calpains in aneurysms of ascending aorta in non-Marfan patients. Therefore, ascending aortic samples of dilated and non-dilated aortas were analyzed according to their calpain-I, -II and calpastatin content as well as the expression levels of MMPs and elastin as well as the infiltration of inflammatory cells. We have found significant differences in calpain-I and calpastatin protein expression and serum levels in patients with aneurysm of the ascending aorta. Furthermore, MMP-1 and MMP-3 expression levels correlate with calpain-I protein levels. Due to our findings we conclude that calpain-1 seems to be related to fibrotic alteration in aortic aneurysm tissue in our experimental group. The change in calpain-1 modulates the structure of aortic tissue causing alteration in elastin structure, thus enabling macrophage infiltration and elevation of MMP levels. Circulating levels of calpain-1 may be used as a prognostic marker in the future if further correlation analyses are done. PMID:26582478

  20. Proteolytic assays on quantum-dot-modified paper substrates using simple optical readout platforms.

    PubMed

    Petryayeva, Eleonora; Algar, W Russ

    2013-09-17

    Paper-based assays are a promising diagnostic format for point-of-care applications, field deployment, and other low-resource settings. To date, the majority of efforts to integrate nanomaterials with paper-based assays have utilized gold nanoparticles. Here, we show that semiconductor quantum dots (QDs), in combination with Förster resonance energy transfer (FRET), are also suitable nanomaterials for developing paper-based assays. Paper fibers were chemically modified with thiol ligands to immobilize CdSeS/ZnS QDs, the QDs were self-assembled with dye-labeled peptides to generate efficient FRET, and steady-state and fluorescence lifetime imaging microscopy (FLIM) were used for characterization. Peptides were selected as substrates for three different proteases and a series of kinetic assays for proteolytic activity was carried out, including multiplexed assays and pro-enzyme activation assays. Quantitative results were obtained within 5-60 min at levels as low as 1-2 nM of protease. These assays were possible using simple optical readout platforms that did not negate the low cost, ease of use, and overall accessibility advantages of paper-based assays. A violet light-emitting diode (LED) excitation source and color imaging with either a digital camera, consumer webcam, or smartphone camera were sufficient for analysis on the basis of a red/green color intensity ratio. At most, a universal serial bus (USB) connection to a computer was required and the instrumentation cost orders of magnitude less than that typically utilized for in vitro bioanalyses with QDs. This work demonstrates that QDs are valuable probes for developing a new generation of paper-based diagnostics. PMID:23980758

  1. Contribution of starter cultures to the proteolytic process of a fermented non-dried whole muscle ham product.

    PubMed

    Scannell, Amalia G M; Kenneally, Paul M; Arendt, Elke K

    2004-06-01

    Porcine longissimus dorsi muscles were cured by brine injection. Curing brine containing 15% (w/v) NaCl, 1.33% (w/v) glucose, 750 ppm sodium nitrite, and appropriate levels of either Lactobacillus sakei LAD, L. sakei LAD plus Kocuria varians FT4 (formally Micrococcus varians), L. sakei LAD plus papain and GDL (glucono-delta-lactone) plus K. varians FT4, was injected to the muscle at a pumping rate 15% w/v. The effect of these treatments on the proteolysis in the ham system was compared to a control ham, produced without starter culture and containing GDL acidulant to control pH and antibiotics to reduce the contribution of background microflora. Hydrolysis of sarcoplasmic and myofibrillar protein fractions was evaluated by SDS-PAGE and reverse phase-HPLC. Hams with different treatments were also investigated for differences in amino acid profile, protein and non-protein nitrogen level, colour, pH, water activity and moisture and microbiological evolution. There was no significant difference in the gross compositional analysis of any of the treatments compared to the control. There was no significant difference (p>0.05) in the protein content, non-protein nitrogen level, SDS-PAGE and free amino acid analysis between the control ham and ham inoculated with proteolytic starter culture. However, it was observed that hams containing starter cultures exhibited decreases in certain peptide fractions and corresponding increases in some free amino acids compared to the uninoculated control. It can be concluded that, while the principle mechanisms resulting in the proteolysis of this non-dried ham product involve the activity of endogeneous cathepsins, the addition of proteolytic starter cultures influence the amino acid profile thereby potentially enhancing the sensorial attributes of the ham. PMID:15135960

  2. Inhibition of proteolytic processing of adenoviral proteins by epsilon-aminocaproic acid and ambenum in adenovirus-infected cells.

    PubMed

    Nosach, Lidiya; Dyachenko, Nataliya; Zhovnovataya, Valentina; Lozinskiy, Miron; Lozitsky, Victor

    2002-01-01

    Maturation of adenovirus particles is markedly affected by proteolytic processing. The possibility for blocking the conversion of precursor structural core protein (preVII) into mature structure protein VII by officinal drugs epsilon-aminocaproic acid and ambenum has been demonstrated in Hep-2 cells infected with adenovirus. Proteolytic processing may be regarded as one of the targets for inhibiting adenovirus reproduction. PMID:12545207

  3. Proteolytic degradation of ewe milk proteins during fermentation of yoghurts and storage.

    PubMed

    El-Zahar, Khaled; Chobert, Jean-Marc; Sitohy, Mahmoud; Dalgalarrondo, Michèle; Haertlé, Thomas

    2003-06-01

    Yoghurts are mostly produced from cow milk and to a very limited extent from ewe milk. The evolution of caseins and whey proteins in ovine milk submitted to different thermal treatments (63 degrees C/30 min; 73 degrees C/15 min; 85 degrees C/10 min or 96 degrees C/5 min) was followed during fermentation of yoghurts and during their storage up to 14 days, using two different sets of starters. One set of starter LAB was a "ropy" culture (YC-191), which is a well-defined mixed strain culture containing Streptococcus thermophilus ST-143 and Lactobacillus delbrueckii subsp. bulgaricus (LB-18 and LB-CH2). The other set of starter bacteria (YC-460) was a standard yoghurt culture("non-ropy") containing mixed strain culture of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. Contents of free amino groups in produced yoghurts increased gradually during the fermentation, up to a maximal value obtained after 4 h fermentation, then they did not change significantly during storage of yoghurt produced with YC-191 starter. In contrary, a large drop in the amount of free amino groups was observed in the first 24 h of storage in the case of yoghurt made with YC-460 indicating that microorganisms continue still to grow in low temperatures. During fermentation and storage of both yoghurt types, alpha-lactalbumin was hydrolyzed to a slightly bigger extent than beta-lactoglobulin. During fermentation, beta-casein was slightly more degraded than alpha(s)-caseins; however, the opposite was observed during storage up to 14 days. Generally, a more intense heat pretreatment led to a higher degradation of whey proteins and caseins during fermentation and storage. Differences in proteolytic activity between the two starters used (whey proteins more degraded by YC-191; caseins more degraded by YC-460) may lead to improvement in production and formulation of yoghurts differing in their physicochemical and rheological properties. PMID:12866624

  4. Splicing and proteolytic processing in VEGF signaling: now it is the coreceptor's turn.

    PubMed

    Yao, Xiaolan; Bouyain, Samuel

    2015-04-01

    Alternative splicing and proteolytic processing of VEGFs generate proteins with distinct physiological roles. In this issue of Structure, Parker et al. show that proteolysis of an isoform of the VEGF-C coreceptor Nrp2 produces a soluble receptor that inhibits VEGF-C/Nrp2 interactions. PMID:25862932

  5. Changes in expression of proteolytic genes in response to anabolic and catabolic signals in rainbow trout

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rates of protein accrual are largely affected by rates of protein degradation. Determining how proteolytic pathways are affected by catabolic and anabolic signals will contribute to the understanding of the impact and regulation these pathways have on protein turnover. Real time RT-PCR was used to...

  6. Site-specific proteolytic degradation of IgG monoclonal antibodies expressed in tobacco plants.

    PubMed

    Hehle, Verena K; Lombardi, Raffaele; van Dolleweerd, Craig J; Paul, Mathew J; Di Micco, Patrizio; Morea, Veronica; Benvenuto, Eugenio; Donini, Marcello; Ma, Julian K-C

    2015-02-01

    Plants are promising hosts for the production of monoclonal antibodies (mAbs). However, proteolytic degradation of antibodies produced both in stable transgenic plants and using transient expression systems is still a major issue for efficient high-yield recombinant protein accumulation. In this work, we have performed a detailed study of the degradation profiles of two human IgG1 mAbs produced in plants: an anti-HIV mAb 2G12 and a tumour-targeting mAb H10. Even though they use different light chains (κ and λ, respectively), the fragmentation pattern of both antibodies was similar. The majority of Ig fragments result from proteolytic degradation, but there are only a limited number of plant proteolytic cleavage events in the immunoglobulin light and heavy chains. All of the cleavage sites identified were in the proximity of interdomain regions and occurred at each interdomain site, with the exception of the VL /CL interface in mAb H10 λ light chain. Cleavage site sequences were analysed, and residue patterns characteristic of proteolytic enzymes substrates were identified. The results of this work help to define common degradation events in plant-produced mAbs and raise the possibility of predicting antibody degradation patterns 'a priori' and designing novel stabilization strategies by site-specific mutagenesis. PMID:25283551

  7. Alleviation of Proteolytic Sensitivity To Enhance Recombinant Lipase Production in Escherichia coli▿

    PubMed Central

    Narayanan, Niju; Chou, C. Perry

    2009-01-01

    Two amino acids, Leu149 and Val223, were identified as proteolytically sensitive when Pseudozyma antarctica lipase (PalB) was heterologously expressed in Escherichia coli. The functional expression was enhanced using the double mutant for cultivation. However, the recombinant protein production was still limited by PalB misfolding, which was resolved by DsbA coexpression. PMID:19542329

  8. Proteolytic processing of Porcine Reproductive and Respiratory Syndrome Virus nsp2 replicase protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    One critical step in porcine reproductive and respiratory syndrome virus (PRRSV) replication is the proteolytic processing of the ORF1 polyprotein (replicase). The replicase polyprotein is generally believed to be processed to generate at least 12 smaller nonstructural proteins (nsps) involved in r...

  9. Proteolytic Products of the Porcine Reproductive and Respiratory Syndrome Virus Nsp2 Replicase Protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The nsp2 replicase protein of porcine reproductive and respiratory syndrome virus (PRRSV) was recently demonstrated to be processed from its precursor by the PL2 protease at or near the G1196|G1197 dipeptide in transfected CHO cells. Here, the proteolytic cleavage of PRRSV nsp2 was further investiga...

  10. Valorization of tomato waste proteins through production of antioxidant and antibacterial hydrolysates by proteolytic Bacillus subtilis: optimization of fermentation conditions.

    PubMed

    Moayedi, Ali; Hashemi, Maryam; Safari, Mohammad

    2016-01-01

    In this study, protein-rich waste of tomato processing industries was fermented by Bacillus subtilis A14h to produce hydrolysates with antioxidant and antibacterial activities. The effects of different levels of initial pH, incubation temperature, fermentation time, protein concentration and inoculum size on proteolytic activity, release of amino acids and peptides, antioxidant and antibacterial activities of hydrolysates were evaluated and optimized by using response surface methodology (RSM). Results showed that all the evaluated variables significantly influenced the hydrolysis and bioactivities of hydrolysates in polynomial models. Hydrolysates showed remarkable 2, 2'-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity (up to 70 %), ferric ion reducing power, and inhibitory activity against B. cereus (up to 69.8 %) and E. coli (up to 29.8 %). Overall, good correlation between the concentration of amino acids and peptides, and antioxidant as well as antibacterial activities (in particular for B. cereus inhibition activity) was observed. Finally, optimum conditions for fermentative conversion of tomato waste proteins to antioxidant and antibacterial hydrolysates were established. Results of this study showed that tomato waste protein can be valorized to produce antioxidant and antibacterial hydrolysates in a fermentative system using B. subtilis A14h. PMID:26787958

  11. Proteolytic Digestion and TiO2 Phosphopeptide Enrichment Microreactor for Fast MS Identification of Proteins.

    PubMed

    Deng, Jingren; Lazar, Iulia M

    2016-04-01

    The characterization of phosphorylation state(s) of a protein is best accomplished by using isolated or enriched phosphoprotein samples or their corresponding phosphopeptides. The process is typically time-consuming as, often, a combination of analytical approaches must be used. To facilitate throughput in the study of phosphoproteins, a microreactor that enables a novel strategy for performing fast proteolytic digestion and selective phosphopeptide enrichment was developed. The microreactor was fabricated using 100 μm i.d. fused-silica capillaries packed with 1-2 mm beds of C18 and/or TiO2 particles. Proteolytic digestion-only, phosphopeptide enrichment-only, and sequential proteolytic digestion/phosphopeptide enrichment microreactors were developed and tested with standard protein mixtures. The protein samples were adsorbed on the C18 particles, quickly digested with a proteolytic enzyme infused over the adsorbed proteins, and further eluted onto the TiO2 microreactor for enrichment in phosphopeptides. A number of parameters were optimized to speed up the digestion and enrichments processes, including microreactor dimensions, sample concentrations, digestion time, flow rates, buffer compositions, and pH. The effective time for the steps of proteolytic digestion and enrichment was less than 5 min. For simple samples, such as standard protein mixtures, this approach provided equivalent or better results than conventional bench-top methods, in terms of both enzymatic digestion and selectivity. Analysis times and reagent costs were reduced ~10- to 15-fold. Preliminary analysis of cell extracts and recombinant proteins indicated the feasibility of integration of these microreactors in more advanced workflows amenable for handling real-world biological samples. Graphical Abstract ᅟ. PMID:26883530

  12. Combinatorial protein engineering of proteolytically resistant mesotrypsin inhibitors as candidates for cancer therapy.

    PubMed

    Cohen, Itay; Kayode, Olumide; Hockla, Alexandra; Sankaran, Banumathi; Radisky, Derek C; Radisky, Evette S; Papo, Niv

    2016-05-15

    Engineered protein therapeutics offer advantages, including strong target affinity, selectivity and low toxicity, but like natural proteins can be susceptible to proteolytic degradation, thereby limiting their effectiveness. A compelling therapeutic target is mesotrypsin, a protease up-regulated with tumour progression, associated with poor prognosis, and implicated in tumour growth and progression of many cancers. However, with its unique capability for cleavage and inactivation of proteinaceous inhibitors, mesotrypsin presents a formidable challenge to the development of biological inhibitors. We used a powerful yeast display platform for directed evolution, employing a novel multi-modal library screening strategy, to engineer the human amyloid precursor protein Kunitz protease inhibitor domain (APPI) simultaneously for increased proteolytic stability, stronger binding affinity and improved selectivity for mesotrypsin inhibition. We identified a triple mutant APPIM17G/I18F/F34V, with a mesotrypsin inhibition constant (Ki) of 89 pM, as the strongest mesotrypsin inhibitor yet reported; this variant displays 1459-fold improved affinity, up to 350 000-fold greater specificity and 83-fold improved proteolytic stability compared with wild-type APPI. We demonstrated that APPIM17G/I18F/F34V acts as a functional inhibitor in cell-based models of mesotrypsin-dependent prostate cancer cellular invasiveness. Additionally, by solving the crystal structure of the APPIM17G/I18F/F34V-mesotrypsin complex, we obtained new insights into the structural and mechanistic basis for improved binding and proteolytic resistance. Our study identifies a promising mesotrypsin inhibitor as a starting point for development of anticancer protein therapeutics and establishes proof-of-principle for a novel library screening approach that will be widely applicable for simultaneously evolving proteolytic stability in tandem with desired functionality for diverse protein scaffolds. PMID:26957636

  13. Enhanced Proteolytic Processing of Recombinant Human Coagulation Factor VIII B-Domain Variants by Recombinant Furins.

    PubMed

    Demasi, Marcos A; de S Molina, Erika; Bowman-Colin, Christian; Lojudice, Fernando H; Muras, Angelita; Sogayar, Mari C

    2016-06-01

    Recombinant human factor VIII (rFVIII) is used in replacement therapy for hemophilia A. Current research efforts are focused on bioengineering rFVIII molecules to improve its secretion efficiency and stability, limiting factors for its efficient production. However, high expression yield in mammalian cells of these rFVIII variants is generally associated with limited proteolytic processing. Non-processed single-chain polypeptides constitute non-natural FVIII molecule configurations with unpredictable toxicity and/or antigenicity. Our main objective was to demonstrate the feasibility of promoting full-proteolytic processing of an rFVIII variant retaining a portion of the B-domain, converting it into the smallest natural activatable form of rFVIII, while keeping its main advantage, i.e., improved secretion efficiency. We generated and employed a CHO-DG44 cell clone producing an rFVIII variant retaining a portion of the B-domain and the FVIII native cleavage site between Arg(1648) and Glu(1649). By bioengineering CHO-DG44 cells to express stably the recombinant human endoproteases PACE, PACE-SOL, PCSK5, PCSK6, or PCKS7, we were able to achieve complete intra- or extracellular proteolytic processing of this rFVIII variant. Additionally, our quantitative data indicated that removal of the B-domain segment by intracellular proteolytic processing does not interfere with this rFVIII variant secretion efficiency. This work also provides the first direct evidence of (1) intracellular cleavage at the Arg(1648) FVIII processing site promoted by wild-type PACE and PCSK7 and (2) proteolytic processing at the Arg(1648) FVIII processing site by PCSK6. PMID:27126696

  14. Proteolytic Digestion and TiO2 Phosphopeptide Enrichment Microreactor for Fast MS Identification of Proteins

    NASA Astrophysics Data System (ADS)

    Deng, Jingren; Lazar, Iulia M.

    2016-04-01

    The characterization of phosphorylation state(s) of a protein is best accomplished by using isolated or enriched phosphoprotein samples or their corresponding phosphopeptides. The process is typically time-consuming as, often, a combination of analytical approaches must be used. To facilitate throughput in the study of phosphoproteins, a microreactor that enables a novel strategy for performing fast proteolytic digestion and selective phosphopeptide enrichment was developed. The microreactor was fabricated using 100 μm i.d. fused-silica capillaries packed with 1-2 mm beds of C18 and/or TiO2 particles. Proteolytic digestion-only, phosphopeptide enrichment-only, and sequential proteolytic digestion/phosphopeptide enrichment microreactors were developed and tested with standard protein mixtures. The protein samples were adsorbed on the C18 particles, quickly digested with a proteolytic enzyme infused over the adsorbed proteins, and further eluted onto the TiO2 microreactor for enrichment in phosphopeptides. A number of parameters were optimized to speed up the digestion and enrichments processes, including microreactor dimensions, sample concentrations, digestion time, flow rates, buffer compositions, and pH. The effective time for the steps of proteolytic digestion and enrichment was less than 5 min. For simple samples, such as standard protein mixtures, this approach provided equivalent or better results than conventional bench-top methods, in terms of both enzymatic digestion and selectivity. Analysis times and reagent costs were reduced ~10- to 15-fold. Preliminary analysis of cell extracts and recombinant proteins indicated the feasibility of integration of these microreactors in more advanced workflows amenable for handling real-world biological samples.

  15. Cell-associated proteolytic enzymes from marine phytoplankton

    SciTech Connect

    Berges, J.A.; Falkowski, P.G.

    1996-08-01

    Despite their central importance in cell metabolism, little is known about proteases in marine phytoplankton. Caseinolytic and leucine aminopeptidase (LAP) activities was surveyed in log-phase cultures of the chlorophyte Dunaliella tertiolecta Butcher, the diatom Thalassiosira weissflogii Fryxell et Hasle, the chrysophyte Isochrysis galbana Parke, the coccolithophorid Emiliania huxleyi Hay et Mohler, and the cyanobacterium Synechococcus sp. LAP activity was very low at pH < 6 and peaked between pH 7.5 and 8.5 in all species, whereas caseinolytic activity in most species showed only minor peaks in the pH 4-5 range and broad maxima above pH 8. Acidic vacuolar proteases apparently represented only a small fraction of total protease activity. Attempts to classify protease using selective inhibitors were inconclusive. Caserinolytic activities were remarkably stable. Casein zymograms were used to identify >200-and <20-kDa proteases in homogenates of log-phase T. weissflogii; only the smaller protease was found in D. tertiolecta. Antibodies in the ATPase subunit (C) of the conserved, chloroplastic Clp protease from Pisum cross-reacted with proteins in Synechococcus, D. tertiolecta, and I. galbana, but no cross-reactions were found for any species with antibodies against the ClpP subunit from either E. coli or Nicotiana. Our results show that phytoplankton contain a diverse complement of proteases with novel characteristics. 46 refs., 6 figs., 1 tab.

  16. [Proteolytic enzymes as markers of productivity and heterosis of silkworm].

    PubMed

    Krylova, I D; Iarygin, D V; Filippovich, Iu B

    2005-01-01

    A positive correlation between the activity level of cysteine proteinases in developing eggs of common silkworm moth (Bombyx mori L.), on the one hand, and a set of commercial characteristics, on the other, was found. This allows the determination of cysteine proteinase activities (pH optima of 3.0, 3.6, and 8.6) to be recommended as a biochemical test for an early prediction of potential productivity of silkworm breeds. A positive correlation between the activity level of acid cysteine proteinases in eggs of parental breeds and a set of commercial characteristics of their hybrids was detected, indicating a principal possibility of predicting the degree of heterosis. PMID:15810740

  17. [A new method of immobilizing proteolytic enzymes in polymeric hydrogels].

    PubMed

    Markvicheva, E A; Bronin, A S; Kudriavtseva, N E; Kuz'kina, I F; Pashkin, I I; Kirsh, Iu E; Rumsh, L D; Zubov, V P

    1994-03-01

    A novel one-step method for enzyme immobilization in composite gels based on the thermally reversible polymer poly (N-vinyl-caprolactam) was developed. Conditions for entrapment of trypsin and carboxypeptidase B in the gels were optimized. After immobilization, 80 and 90% of the original carboxypeptidase B and trypsin activities, respectively, were found to retain. Both immobilizes enzymes were active in a wider pH and temperature range. The granules with entrapped enzymes were successfully applied to obtain human insulin from recombinant proinsulin. PMID:8166751

  18. Proteolytic inactivation of the leukocyte C5a receptor by proteinases derived from Porphyromonas gingivalis.

    PubMed Central

    Jagels, M A; Travis, J; Potempa, J; Pike, R; Hugli, T E

    1996-01-01

    The anaerobic bacterium Porphyromonas gingivalis has been implicated as a primary causative agent in adult periodontitis. Several proteinases are produced by this bacterium, and it is suggested that they contribute to virulence and to local tissue injury resulting from infection by P. gingivalis. Cysteine proteinases with specificities to cleave either Arg-X or Lys-X peptide bonds (i.e., gingipains) have been characterized as predominant enzymes associated with vesicles shed from the surface of this bacterium. It has recently been demonstrated that these proteinases are capable of degrading the blood complement component C5, resulting in the generation of biologically active C5a. By using an affinity-purified rabbit antibody raised against residues 9 to 29 of the C5a receptor (C5aR; CD88), we demonstrate that noncysteinyl proteinases associated with vesicles obtained from P. gingivalis cleave the C5aR on human neutrophils. Proteolytic attack of the C5aR by enzymes from the P. gingivalis vesicles was inhibited by TPCK (tolylsullonyl phenylalanyl chloromethyl ketone), PMSF (phenylmethylsulfonyl fluoride), and dichloroisocoumarin, suggesting that serine proteinases are primarily responsible for this degradative activity. The purified vesicle proteinase Lys-gingipain but not Arg-gingipain also cleaved the N-terminal region of the C5aR on the human neutrophils. Lys-gingipain activity was essentially resistant to these inhibitors but was inhibited by TLCK (Nalpha-p-tosyl-L-lysine chloromethyl ketone) and iodoacetamide. A synthetic peptide that mimics the N-terminal region of C5aR (residues 9 to 29; PDYGHY DDKDTLDLNTPVDKT) was readily cleaved by chymotrypsin but not by trypsin, despite the presence of two potential trypsin (i.e., lysyl-X) cleavage sites. The specific sites of cleavage in the C5aR 9-29 peptide were determined by mass spectroscopy for both chymotrypsin and Lys-gingipain digests. This analysis demonstrated that the C5aR peptide is susceptible to cleavage at

  19. Properties of proteolytic toxin of Vibrio anguilolarum from diseased flounder

    NASA Astrophysics Data System (ADS)

    Mo, Zhao-Lan; Chen, Shi-Yong; Zhang, Pei-Jun

    2002-12-01

    Extracellular products (ECP) produced by Vibrio anguillarum strain M3 originally isolated from diseased flounder ( Paralichthys olivaceus) were prepared. ECP of M3 showed gelatinase, casinase, amylase and haemolytic activity on agarose plates. High protease activity against azocasin was detected. Bacterium M2 showed highest growth and protease activity at 25°C. The protease present in ECP showed maximal activity at pH 8 and 55°C; was completely inactivated by application of 80°C heat for 30 min; was completely inhibited by EDTA and HgCl2, and was partially inhibited by PMSF, SDS, MnCl2 and iodoacetic acid; but not inhibited by CaCl2 and MgCl2. The ECP was toxic to flounder fish at LD50 values of 3.1 μg protein/g body weight. The addition of HgCl2 and application of heat at 50°C decreased the lethal toxicity of ECP. When heated at 100°C, ECP lethality to flounder was completely inhibited. After intramuscular injection of ECP into flounder, it showed evident histopathological changes including necrosis of muscle, extensive deposition of haemosiderin in the spleen, dilated blood vessels congested with numerious lymphocytes in the liver. These results showed that ECP protease was a lethal factor produced by the bacterium V. anguillarum M3.

  20. The choreography of HIV-1 proteolytic processing and virion assembly.

    PubMed

    Lee, Sook-Kyung; Potempa, Marc; Swanstrom, Ronald

    2012-11-30

    HIV-1 has been the target of intensive research at the molecular and biochemical levels for >25 years. Collectively, this work has led to a detailed understanding of viral replication and the development of 24 approved drugs that have five different targets on various viral proteins and one cellular target (CCR5). Although most drugs target viral enzymatic activities, our detailed knowledge of so much of the viral life cycle is leading us into other types of inhibitors that can block or disrupt protein-protein interactions. Viruses have compact genomes and employ a strategy of using a small number of proteins that can form repeating structures to enclose space (i.e. condensing the viral genome inside of a protein shell), thus minimizing the need for a large protein coding capacity. This creates a relatively small number of critical protein-protein interactions that are essential for viral replication. For HIV-1, the Gag protein has the role of a polyprotein precursor that contains all of the structural proteins of the virion: matrix, capsid, spacer peptide 1, nucleocapsid, spacer peptide 2, and p6 (which contains protein-binding domains that interact with host proteins during budding). Similarly, the Gag-Pro-Pol precursor encodes most of the Gag protein but now includes the viral enzymes: protease, reverse transcriptase (with its associated RNase H activity), and integrase. Gag and Gag-Pro-Pol are the substrates of the viral protease, which is responsible for cleaving these precursors into their mature and fully active forms (see Fig. 1A). PMID:23043111

  1. Evolution of proteolytic and physico-chemical characteristics of Norwegian dry-cured ham during its processing.

    PubMed

    Petrova, Inna; Tolstorebrov, Ignat; Mora, Leticia; Toldrá, Fidel; Eikevik, Trygve Magne

    2016-11-01

    Proteolytic activity and physico-chemical characteristics were studied for Norwegian dry-cured ham at four different times of processing: raw hams, post-salted hams (3 months of processing), hams selected in the middle of the production (12 months of processing) and hams at the end of the processing (24 months). Cathepsin H activity decreased until negligible values after 3 months of processing, whereas cathepsins B and B+L were inactive at 12 months. AAP was the most active aminopeptidase whereas RAP and MAP were active just during the first 12 months of processing. Proteolysis index reached a value of 4.56±1.03 % with non-significant differences between 12 and 24 months of ripening. Peptide identification by LC-MS/MS was done and two peptides (GVEEPPKGHKGNKK and QAISNNKDQGSY) showing a linear response with the time of processing were found. Unfreezable water content and glass transition temperature were investigated using differential scanning calorimetry (DSC) technique with non-significant differences in the temperature of glass transition for 12 and 24 months of processing. PMID:27371871

  2. Loss of proteolytically processed filaggrin caused by epidermal deletion of Matriptase/MT-SP1

    PubMed Central

    List, Karin; Szabo, Roman; Wertz, Philip W.; Segre, Julie; Haudenschild, Christian C.; Kim, Soo-Youl; Bugge, Thomas H.

    2003-01-01

    Profilaggrin is a large epidermal polyprotein that is proteolytically processed during keratinocyte differentiation to release multiple filaggrin monomer units as well as a calcium-binding regulatory NH2-terminal filaggrin S-100 protein. We show that epidermal deficiency of the transmembrane serine protease Matriptase/MT-SP1 perturbs lipid matrix formation, cornified envelope morphogenesis, and stratum corneum desquamation. Surprisingly, proteomic analysis of Matriptase/MT-SP1–deficient epidermis revealed the selective loss of both proteolytically processed filaggrin monomer units and the NH2-terminal filaggrin S-100 regulatory protein. This was associated with a profound accumulation of profilaggrin and aberrant profilaggrin-processing products in the stratum corneum. The data identify keratinocyte Matriptase/MT-SP1 as an essential component of the profilaggrin-processing pathway and a key regulator of terminal epidermal differentiation. PMID:14638864

  3. Role of proteolytic enzymes in degradation of plant tissues

    SciTech Connect

    Lewosz, J.; Kelman, A.; Sequeira, L.

    1991-01-01

    Strain SR 394 of Erwinia carotovora (Ecc) produced proteases constitutively in all media tested. Growth of Ecc and production of protease were enhanced significantly by the presence of poetic materials and/or plant call walls in the test media. After electrofocusing, one major and one minor protease bands, at PI 4.8 and PI 5.1, respectively, were detected. Only one band of 43 kDa was detected on SDS gels. Only one protease band was detected in SDS gels of infected plant extracts. This protease was purified to homogeneity. It in a highly thermostable metal protease; it degrades gelatin, soluble collagen and hide powderazure, shows weak activity on casein and azocasein, but does not degrade insoluble collagen or elastin.

  4. The proteolytic action of Arvin on human fibrinogen

    PubMed Central

    Ewart, M. R.; Hatton, M. W. C.; Basford, J. M.; Dodgson, K. S.

    1970-01-01

    1. Human fibrinogen was subjected to proteolysis by enzyme preparations (clinical Arvin and IRC-50 Arvin) from the venom of Agkistrodon rhodostoma. 2. IRC-50 Arvin releases three peptides from fibrinogen, and these were identified as fibrinopeptides AP, AY and A. 3. The less purified `clinical' Arvin releases, in addition to fibrinopeptides AP, AY and A, small amounts of two heptapeptides derived from fibrinopeptides AP and A, probably because it contains another enzyme as well as Arvin. 4. No fibrinopeptide B is released by either Arvin preparation. 5. Thus, although Arvin is known to differ from `reptilase' from Bothrops jararaca in that it does not activate the enzyme that cross-links fibrin (fibrin-stabilizing factor), it is identical with reptilase with respect to the peptides that it liberates from fibrinogen. PMID:5529716

  5. Autodigestion: Proteolytic Degradation and Multiple Organ Failure in Shock.

    PubMed

    Altshuler, Angelina E; Kistler, Erik B; Schmid-Schönbein, Geert W

    2016-05-01

    There is currently no effective treatment for multiorgan failure following shock other than supportive care. A better understanding of the pathogenesis of these sequelae to shock is required. The intestine plays a central role in multiorgan failure. It was previously suggested that bacteria and their toxins are responsible for the organ failure seen in circulatory shock, but clinical trials in septic patients have not confirmed this hypothesis. Instead, we review here evidence that the digestive enzymes, synthesized in the pancreas and discharged into the small intestine as requirement for normal digestion, may play a role in multiorgan failure. These powerful enzymes are nonspecific, highly concentrated, and fully activated in the lumen of the intestine. During normal digestion they are compartmentalized in the lumen of the intestine by the mucosal epithelial barrier. However, if this barrier becomes permeable, e.g. in an ischemic state, the digestive enzymes escape into the wall of the intestine. They digest tissues in the mucosa and generate small molecular weight cytotoxic fragments such as unbound free fatty acids. Digestive enzymes may also escape into the systemic circulation and activate other degrading proteases. These proteases have the ability to clip the ectodomain of surface receptors and compromise their function, for example cleaving the insulin receptor causing insulin resistance. The combination of digestive enzymes and cytotoxic fragments leaking into the central circulation causes cell and organ dysfunction, and ultimately may lead to complete organ failure and death. We summarize current evidence suggesting that enteral blockade of digestive enzymes inside the lumen of the intestine may serve to reduce acute cell and organ damage and improve survival in experimental shock. PMID:26717111

  6. N-Terminal Enrichment: Developing a Protocol to Detect Specific Proteolytic Fragments

    PubMed Central

    Schepmoes, Athena A.; Zhang, Qibin; Petritis, Brianne O.; Qian, Wei-Jun; Smith, Richard D.

    2009-01-01

    Proteolytic processing events are essential to physiological processes such as reproduction, development, and host responses, as well as regulating proteins in cancer; therefore, there is a significant need to develop robust approaches for characterizing such events. The current mass spectrometry (MS)-based proteomics techniques employs a “bottom-up” strategy, which does not allow for identification of different proteolytic proteins since the strategy measures all the small peptides from any given protein. The aim of this development is to enable the effective identification of specific proteolytic fragments. The protocol utilizes an acetylation reaction to block the N-termini of a protein, as well as any lysine residues. Following digestion, N-terminal peptides are enriched by removing peptides that contain free amines, using amine-reactive silica-bond succinic anhydride beads. The resulting enriched sample has one N-terminal peptide per protein, which reduces sample complexity and allows for increased analytical sensitivity compared to global proteomics.1 We initially compared the peptide identification and efficiency of blocking lysine using acetic anhydride (a 42 Da modification) or propionic anhydride (a 56 Da modification) in our protocol. Both chemical reactions resulted in comparable peptide identifications and ∼95 percent efficiency for blocking lysine residues. However, the use of propionic anhydride allowed us to distinguish in vivo acetylated peptides from chemically-tagged peptides.2 In an initial experiment using mouse plasma, we were able to identify >300 unique N-termini peptides, as well as many known cleavage sites. This protocol holds potential for uncovering new information related to proteolytic pathways, which will assist our understanding about cancer biology and efforts to identify potential biomarkers for various diseases. PMID:19949699

  7. Independent evolution of neurotoxin and flagellar genetic loci in proteolytic Clostridium botulinum

    PubMed Central

    Carter, Andrew T; Paul, Catherine J; Mason, David R; Twine, Susan M; Alston, Mark J; Logan, Susan M; Austin, John W; Peck, Michael W

    2009-01-01

    Background Proteolytic Clostridium botulinum is the causative agent of botulism, a severe neuroparalytic illness. Given the severity of botulism, surprisingly little is known of the population structure, biology, phylogeny or evolution of C. botulinum. The recent determination of the genome sequence of C. botulinum has allowed comparative genomic indexing using a DNA microarray. Results Whole genome microarray analysis revealed that 63% of the coding sequences (CDSs) present in reference strain ATCC 3502 were common to all 61 widely-representative strains of proteolytic C. botulinum and the closely related C. sporogenes tested. This indicates a relatively stable genome. There was, however, evidence for recombination and genetic exchange, in particular within the neurotoxin gene and cluster (including transfer of neurotoxin genes to C. sporogenes), and the flagellar glycosylation island (FGI). These two loci appear to have evolved independently from each other, and from the remainder of the genetic complement. A number of strains were atypical; for example, while 10 out of 14 strains that formed type A1 toxin gave almost identical profiles in whole genome, neurotoxin cluster and FGI analyses, the other four strains showed divergent properties. Furthermore, a new neurotoxin sub-type (A5) has been discovered in strains from heroin-associated wound botulism cases. For the first time, differences in glycosylation profiles of the flagella could be linked to differences in the gene content of the FGI. Conclusion Proteolytic C. botulinum has a stable genome backbone containing specific regions of genetic heterogeneity. These include the neurotoxin gene cluster and the FGI, each having evolved independently of each other and the remainder of the genetic complement. Analysis of these genetic components provides a high degree of discrimination of strains of proteolytic C. botulinum, and is suitable for clinical and forensic investigations of botulism outbreaks. PMID:19298644

  8. N-Terminal Enrichment: Developing a Protocol to Detect Specific Proteolytic Fragments

    SciTech Connect

    Schepmoes, Athena A.; Zhang, Qibin; Petritis, Brianne O.; Qian, Weijun; Smith, Richard D.

    2009-12-01

    Proteolytic processing events are essential to physiological processes such as reproduction, development, and host responses, as well as regulating proteins in cancer; therefore, there is a significant need to develop robust approaches for characterizing such events. The current mass spectrometry (MS)-based proteomics techniques employs a “bottom-up” strategy, which does not allow for identification of different proteolytic proteins since the strategy measures all the small peptides from any given protein. The aim of this development is to enable the effective identification of specific proteolytic fragments. The protocol utilizes an acetylation reaction to block the N-termini of a protein, as well as any lysine residues. Following digestion, N-terminal peptides are enriched by removing peptides that contain free amines, using amine-reactive silica-bond succinic anhydride beads. The resulting enriched sample has one N-terminal peptide per protein, which reduces sample complexity and allows for increased analytical sensitivity compared to global proteomics.1 We initially compared the peptide identification and efficiency of blocking lysine using acetic anhydride (a 42 Da modification) or propionic anhydride (a 56 Da modification) in our protocol. Both chemical reactions resulted in comparable peptide identifications and *95 percent efficiency for blocking lysine residues. However, the use of propionic anhydride allowed us to distinguish in vivo acetylated peptides from chemically-tagged peptides.2 In an initial experiment using mouse plasma, we were able to identify *300 unique N-termini peptides, as well as many known cleavage sites. This protocol holds potential for uncovering new information related to proteolytic pathways, which will assist our understanding about cancer biology and efforts to identify potential biomarkers for various diseases.

  9. On the agglutinogens of red cells developed with proteolytic enzymes and neuraminidase.

    PubMed

    Sagisaka, K; Takahashi, K

    1976-10-01

    It has been known that the agglutinability of human red cells is changed or enhanced by treatments with proteolytic enzymes or neuraminidase. In this paper, the serological properties of agglutinogens developed by proteolytic enzymes (bromelin, ficin, papain, trypsin and pronase) and neuraminidase are investigated by using antisera to trypsin- and neuraminidase-treated red cells. The adsorptions of the antiserum to trypsinized red cells with the cells treated with each of the proteolytic enzymes showed that the agglutinogens uncovered by bromelin, ficin and papain were different from those by pronase and trypsin. It was demonstrated that pronase was the most effective enzyme to uncover the agglutinogen located on deeper site of red cell membrane. This was confirmed by the agglutination with the test cells treated twice with two kinds of the enzymes. The reactions of the antiserum to neuraminidase-treated red cells treated with six kinds of the enzymes indicated that the agglutinogens developed by neuraminidase resembled those by bromelin, ficin and papain more than those by trypsin and pronase. PMID:982434

  10. Analyzing Protease Specificity and Detecting in Vivo Proteolytic Events Using Tandem Mass Spectrometry

    SciTech Connect

    Gupta, Nitin; Hixson, Kim K.; Culley, David E.; Smith, Richard D.; Pevzner, Pavel A.

    2010-07-01

    While trypsin remains the most commonly used protease in mass spectrometry, other proteases may be employed for increasing peptide-coverage or generating overlapping peptides. Knowledge of the accurate specifcity rules of these proteases is helpful for database search tools to detect peptides, and becomes crucial when mass spectrometry is used to discover in vivo proteolytic cleavages. In this study, we use tandem mass spectrometry to analyze the specifcity rules of selected proteases and describe MS- Proteolysis, a software tool for identifying putative sites of in vivo proteolytic cleavage. Our analysis suggests that the specifcity rules for some commonly used proteases can be improved, e.g., we find that V8 protease cuts not only after Asp and Glu, as currently expected, but also shows a smaller propensity to cleave after Gly for the conditions tested in this study. Finally, we show that comparative analysis of multiple proteases can be used to detect putative in vivo proteolytic sites on a proteome-wide scale.

  11. Leucoagglutinating phytohemagglutinin: purification, characterization, proteolytic digestion and assessment for allergenicity potential in BALB/c mice.

    PubMed

    Kumar, Sandeep; Sharma, Akanksha; Das, Mukul; Jain, S K; Dwivedi, Premendra D

    2014-04-01

    Red kidney bean (Phaseolus vulgaris) is consumed worldwide as a vegetarian protein source. But, at the same time the allergenicity potential of red kidney bean is a matter of concern. This study is aimed towards purification, characterization, thermal stability, proteolytic digestion and allergenicity assessment of one of the clinically relevant allergens of red kidney bean. The purification of red kidney bean allergic protein was carried out with the help of column chromatography, IgE immunoblotting and reverse phase high-pressure liquid chromatography (RP-HPLC). The purified protein was characterized by peptide mass finger printing (PMF) and studied for its thermal stability, and proteolytic resistance using simulated gastric fluid (SGF) assay. The allergenicity potential of the purified protein was studied in BALB/c mice. The purified protein was identified as leucoagglutinating phytohemagglutinin (PHA-L) with molecular weight 29.5 kDa. The PHA-L showed resistance to heat as well as proteolytic enzyme. Higher levels of total IgE, specific IgE, and histamine were observed in PHA-L treated BALB/c mice when compared to control. Overall, PHA-L possesses characteristics of allergens and may play a potential role in the red kidney bean induced allergy. PMID:24548135

  12. Detection of the apr gene in proteolytic psychrotrophic bacteria isolated from refrigerated raw milk.

    PubMed

    Martins, Maurilio L; de Araújo, Elza F; Mantovani, Hilário C; Moraes, Célia A; Vanetti, Maria C D

    2005-07-15

    Bacteria of the genus Pseudomonas have been associated with the spoilage of raw milk and dairy products due to the production of thermostable proteolytic enzymes. The apr gene encodes for alkaline metalloprotease in Pseudomonas and other related bacteria. Its presence in psychrotrophic proteolytic bacteria isolated from raw milk collected from cooling tanks was verified. A polymerase chain reaction (PCR) technique was used with degenerate primers. Total DNA from 112 isolates was pooled in different groups and then used as template for the amplification reactions. Controls consisted of DNA extracted from 26 cultures. An expected DNA fragment of 194 bp was detected in groups that contained bacteria identified as Pseudomonas. The PCR product was observed only when DNA from control cultures of Pseudomonas aeruginosa, Pseudomonas fluorescens, Serratia marcescens and Aeromonas hydrophila were used. A detection limit assay indicated that the apr gene could be directly amplified from pasteurized milk inoculated with 10(8) CFU/ml of P. fluorescens. With this method it was possible to detect proteolytic bacteria at 10(5) CFU/ml in reconstituted skim milk powder if cells were recovered for DNA extraction before amplification. PMID:15992619

  13. Coronavirus and Influenza Virus Proteolytic Priming Takes Place in Tetraspanin-Enriched Membrane Microdomains

    PubMed Central

    Earnest, James T.; Hantak, Michael P.; Park, Jung-Eun

    2015-01-01

    ABSTRACT Coronaviruses (CoVs) and low-pathogenicity influenza A viruses (LP IAVs) depend on target cell proteases to cleave their viral glycoproteins and prime them for virus-cell membrane fusion. Several proteases cluster into tetraspanin-enriched microdomains (TEMs), suggesting that TEMs are preferred virus entry portals. Here we found that several CoV receptors and virus-priming proteases were indeed present in TEMs. Isolated TEMs, when mixed with CoV and LP IAV pseudoparticles, cleaved viral fusion proteins to fusion-primed fragments and potentiated viral transductions. That entering viruses utilize TEMs as a protease source was further confirmed using tetraspanin antibodies and tetraspanin short hairpin RNAs (shRNAs). Tetraspanin antibodies inhibited CoV and LP IAV infections, but their virus-blocking activities were overcome by expressing excess TEM-associated proteases. Similarly, cells with reduced levels of the tetraspanin CD9 resisted CoV pseudoparticle transductions but were made susceptible by overproducing TEM-associated proteases. These findings indicated that antibodies and CD9 depletions interfere with viral proteolytic priming in ways that are overcome by surplus proteases. TEMs appear to be exploited by some CoVs and LP IAVs for appropriate coengagement with cell receptors and proteases. IMPORTANCE Enveloped viruses use their surface glycoproteins to catalyze membrane fusion, an essential cell entry step. Host cell components prime these viral surface glycoproteins to catalyze membrane fusion at specific times and places during virus cell entry. Among these priming components are proteases, which cleave viral surface glycoproteins, unleashing them to refold in ways that catalyze virus-cell membrane fusions. For some enveloped viruses, these proteases are known to reside on target cell surfaces. This research focuses on coronavirus and influenza A virus cell entry and identifies TEMs as sites of viral proteolysis, thereby defining subcellular

  14. The Alteration of Plant Morphology by Small Peptides Released from the Proteolytic Processing of the Bacterial Peptide TENGU1[W

    PubMed Central

    Sugawara, Kyoko; Honma, Youhei; Komatsu, Ken; Himeno, Misako; Oshima, Kenro; Namba, Shigetou

    2013-01-01

    Phytoplasmas are insect-borne plant pathogenic bacteria that alter host morphology. TENGU, a small peptide of 38 residues, is a virulence factor secreted by phytoplasmas that induces dwarfism and witches’ broom in the host plant. In this study, we demonstrate that plants process TENGU in order to generate small functional peptides. First, virus vector-mediated transient expression demonstrated that the amino-terminal 11 amino acids of TENGU are capable of causing symptom development in Nicotiana benthamiana plants. The deletion of the 11th residue significantly diminished the symptom-inducing activity of TENGU, suggesting that these 11 amino acids constitute a functional domain. Second, we found that TENGU undergoes proteolytic processing in vitro, generating peptides of 19 and 21 residues including the functional domain. Third, we observed similar processing of TENGU in planta, and an alanine substitution mutant of TENGU, for which processing was compromised, showed reduced symptom induction activity. All TENGU homologs from several phytoplasma strains possessed similar symptom induction activity and went through processing, which suggests that the processing of TENGU might be related to its function. PMID:23784461

  15. Oxidative and proteolytic profiles of the right and left heart in a model of cancer-induced cardiac cachexia.

    PubMed

    Borges, F H; Marinello, P C; Cecchini, A L; Blegniski, F P; Guarnier, F A; Cecchini, R

    2014-11-01

    Cardiac cachexia is a syndrome that has received increased attention in recent years. Although an association between proteolysis and cardiac cachexia has been proposed, the direct influence of oxidative stress on the process has not been demonstrated. In the present study, the right (RH) and left (LH) hearts (atrium and ventricle of each side of the heart) were collected from rats at the 5th and 10th days after phosphate buffer (control) orWalker-256 solid tumour implantation. Immediately after sacrifice, cachexia was determined in tumour-bearing animals by the formula: [(inicial body weight-final body weight+tumour weight+weight gain of control group)/(initial body weight+body mass gain of control group)]×100%; RH and LH were stored until use. Oxidative stress and proteolysis were determined in each collected sample. In addition, heart samples were collected from a separate set of animals to determine the thickness of the left and right ventricles. Cachexia values increased over time after tumour implantation from 6.85% at the 5th day to 17.76% at the 10th day. There was no significant difference in LH wet weight and ventricle thickness compared with the control, where as RH wet weight (0.109±0.09g at the 5th day and 0.093±0.09g at the 10th day) and thickness (420±16μm at the 5th day and 279±08μm at the 10th day) were significantly decreased at both time points when compared with control values (0.153±0.06g and 607±21μm, respectively). tert-Butyl-stimulated chemiluminescence analysis revealed a significant increase in the LH and decrease in the RH oxidative stress profiles. Carbonylated proteins increased in the LH (140%, p<0.05) and RH (100%, p<0.05) at the 5th day, and significantly decreased in both sides on the 10th day compared to controls. Chemotrypsin-like, caspase-like, and calpain-like activities were evaluated by chemiluminescence, and only calpain-like activity was found to increase at the 5th day in the RH. In the LH, all proteolytic

  16. Novel Apoptosis Suppressor Apsup from the Baculovirus Lymantria dispar Multiple Nucleopolyhedrovirus Precludes Apoptosis by Preventing Proteolytic Processing of Initiator Caspase Dronc

    PubMed Central

    Yamada, Hayato; Kitaguchi, Koji; Hamajima, Rina; Kobayashi, Michihiro

    2013-01-01

    We previously identified a novel baculovirus-encoded apoptosis suppressor, Apsup, from the baculovirus Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV). Apsup inhibits the apoptosis of L. dispar Ld652Y cells triggered by infection with p35-defective Autographa californica MNPV (vAcΔp35) and exposure to actinomycin D or UV light. Here, we examined the functional role of Apsup in apoptosis regulation in insect cells. Apsup prevented apoptosis and the proteolytic processing of L. dispar initiator caspase Dronc (Ld-Dronc) in Ld652Y cells triggered by overexpression of Ld-Dronc, LdMNPV inhibitor-of-apoptosis 3 (IAP3), or Hyphantria cunea MNPV IAP1. In vAcΔp35-infected apoptotic Ld652Y cells, Apsup restricted apoptosis induction and prevented processing of endogenous Ld-Dronc. Conversely, upon RNA interference (RNAi)-mediated silencing of apsup, LdMNPV-infected Ld652Y cells, which typically support high-titer virus replication, underwent apoptosis, accompanied by the processing of endogenous Ld-Dronc. Furthermore, endogenous Ld-Dronc coimmunoprecipitated with transiently expressed Apsup, indicating that Apsup physically interacts with Ld-Dronc. Apsup prevented the apoptosis of Sf9 cells triggered by vAcΔp35 infection but did not inhibit apoptosis or activation of caspase-3-like protease in vAcΔp35-infected Drosophila melanogaster S2 cells. Apsup also inhibited the proteolytic processing of L. dispar effector caspase Ld-caspase-1 in the transient expression assay but did not physically interact with Ld-caspase-1. These results demonstrate that Apsup inhibits apoptosis in Ld652Y cells by preventing the proteolytic processing of Ld-Dronc. Together with our previous findings showing that Apsup prevents the processing of both overexpressed Ld-Dronc and Bombyx mori Dronc, these results also demonstrate that Apsup functions as an effective apoptotic suppressor in various lepidopteran, but not dipteran, insect cells. PMID:24067961

  17. Contribution of the autophagy-lysosomal and ubiquitin-proteasomal proteolytic systems to total proteolysis in rainbow trout (Oncorhynchus mykiss) myotubes.

    PubMed

    Seiliez, Iban; Dias, Karine; Cleveland, Beth M

    2014-12-01

    The ubiquitin-proteasome system (UPS) is recognized as the major contributor to total proteolysis in mammalian skeletal muscle, responsible for 50% or more of total protein degradation in skeletal muscle, whereas the autophagic-lysosome system (ALS) plays a more minor role. While the relative contribution of these systems to muscle loss is well documented in mammals, little is known in fish species. The current study uses myotubes derived from rainbow trout myogenic precursor cells as an in vitro model of white muscle tissue. Cells were incubated in complete or serum-deprived media or media supplemented with insulin-like growth factor-1 (IGF-1) and exposed to selective proteolytic inhibitors to determine the relative contribution of the ALS and UPS to total protein degradation in myotubes in different culture conditions. Results indicate that the ALS is responsible for 30-34% and 50% of total protein degradation in myotubes in complete and serum-deprived media, respectively. The UPS appears to contribute much less to total protein degradation at almost 4% in cells in complete media to nearly 17% in serum-deprived cells. IGF-1 decreases activity of both systems, as it inhibited the upregulation of both proteolytic systems induced by serum deprivation. The combined inhibition of both the ALS and UPS reduced degradation by a maximum of 55% in serum-deprived cells, suggesting an important contribution of other proteolytic systems to total protein degradation. Collectively, these data identify the ALS as a potential target for strategies aimed at improving muscle protein retention and fillet yield through reductions in protein degradation. PMID:25274907

  18. Neprilysin Inhibits Coagulation through Proteolytic Inactivation of Fibrinogen.

    PubMed

    Burrell, Matthew; Henderson, Simon J; Ravnefjord, Anna; Schweikart, Fritz; Fowler, Susan B; Witt, Susanne; Hansson, Kenny M; Webster, Carl I

    2016-01-01

    Neprilysin (NEP) is an endogenous protease that degrades a wide range of peptides including amyloid beta (Aβ), the main pathological component of Alzheimer's disease (AD). We have engineered NEP as a potential therapeutic for AD but found in pre-clinical safety testing that this variant increased prothrombin time (PT) and activated partial thromboplastin time (APTT). The objective of the current study was to investigate the effect of wild type NEP and the engineered variant on coagulation and define the mechanism by which this effect is mediated. PT and APTT were measured in cynomolgus monkeys and rats dosed with a human serum albumin fusion with an engineered variant of NEP (HSA-NEPv) as well as in control plasma spiked with wild type or variant enzyme. The coagulation factor targeted by NEP was determined using in vitro prothrombinase, calibrated automated thrombogram (CAT) and fibrin formation assays as well as N-terminal sequencing of fibrinogen treated with the enzyme. We demonstrate that HSA-NEP wild type and HSA-NEPv unexpectedly impaired coagulation, increasing PT and APTT in plasma samples and abolishing fibrin formation from fibrinogen. This effect was mediated through cleavage of the N-termini of the Aα- and Bβ-chains of fibrinogen thereby significantly impairing initiation of fibrin formation by thrombin. Fibrinogen has therefore been identified for the first time as a substrate for NEP wild type suggesting that the enzyme may have a role in regulating fibrin formation. Reductions in NEP levels observed in AD and cerebral amyloid angiopathy may contribute to neurovascular degeneration observed in these conditions. PMID:27437944

  19. Neprilysin Inhibits Coagulation through Proteolytic Inactivation of Fibrinogen

    PubMed Central

    Burrell, Matthew; Henderson, Simon J.; Ravnefjord, Anna; Schweikart, Fritz; Fowler, Susan B.; Witt, Susanne; Hansson, Kenny M.; Webster, Carl I.

    2016-01-01

    Neprilysin (NEP) is an endogenous protease that degrades a wide range of peptides including amyloid beta (Aβ), the main pathological component of Alzheimer’s disease (AD). We have engineered NEP as a potential therapeutic for AD but found in pre-clinical safety testing that this variant increased prothrombin time (PT) and activated partial thromboplastin time (APTT). The objective of the current study was to investigate the effect of wild type NEP and the engineered variant on coagulation and define the mechanism by which this effect is mediated. PT and APTT were measured in cynomolgus monkeys and rats dosed with a human serum albumin fusion with an engineered variant of NEP (HSA-NEPv) as well as in control plasma spiked with wild type or variant enzyme. The coagulation factor targeted by NEP was determined using in vitro prothrombinase, calibrated automated thrombogram (CAT) and fibrin formation assays as well as N-terminal sequencing of fibrinogen treated with the enzyme. We demonstrate that HSA-NEP wild type and HSA-NEPv unexpectedly impaired coagulation, increasing PT and APTT in plasma samples and abolishing fibrin formation from fibrinogen. This effect was mediated through cleavage of the N-termini of the Aα- and Bβ-chains of fibrinogen thereby significantly impairing initiation of fibrin formation by thrombin. Fibrinogen has therefore been identified for the first time as a substrate for NEP wild type suggesting that the enzyme may have a role in regulating fibrin formation. Reductions in NEP levels observed in AD and cerebral amyloid angiopathy may contribute to neurovascular degeneration observed in these conditions. PMID:27437944

  20. Proteolytic Processing of Angiotensin-I in Human Blood Plasma

    PubMed Central

    Hildebrand, Diana; Merkel, Philipp; Eggers, Lars Florian; Schlüter, Hartmut

    2013-01-01

    In mammalian species, except humans, N-terminal processing of the precursor peptide angiotensin I (ANG-1-10) into ANG-2-10 or ANG-3-10 was reported. Here we hypothesize that aminopeptidase-generated angiotensins bearing the same C-terminus as ANG-1-10 are also present in humans. We demonstrate the time dependent generation of ANG-2-10, ANG-3-10, ANG-4-10, ANG-5-10 and ANG-6-10 from the precursor ANG-1-10 by human plasma proteins. The endogenous presence of ANG-4-10, ANG-5-10 and ANG-6-10 in human plasma was confirmed by an immuno-fluorescence assay. Generation of ANG-2-10, ANG-3-10 and ANG-4-10 from ANG-1-10 by immobilized human plasma proteins was sensitive to the cysteine/serine protease inhibitor antipain. The metal ion chelator EDTA inhibited Ang-6-10-generation. Incubation of the substrates ANG-3-10, ANG-4-10 and ANG-5-10 with recombinant aminopeptidase N (APN) resulted in a successive N-terminal processing, finally releasing ANG-6-10 as a stable end product, demonstrating a high similarity concerning the processing pattern of the angiotensin peptides compared to the angiotensin generating activity in plasma. Recombinant ACE-1 hydrolyzed the peptides ANG-2-10, ANG-3-10, ANG-4-10 and ANG-5-10 into ANG-2-8, ANG-3-8, ANG-4-8 and ANG-5-8. Since ANG-2-10 was processed into ANG-2-8, ANG-4-8 and ANG-5-8 by plasma proteases the angiotensin peptides bearing the same C-terminus as ANG-1-10 likely have a precursor function in human plasma. Our results confirm the hypothesis of aminopeptidase mediated processing of ANG-1-10 in humans. We show the existence of an aminopeptidase mediated pathway in humans that bypasses the known ANG-1-8-carboxypeptidase pathway. This expands the knowledge about the known human renin angiotensin system, showing how efficiently the precursor ANG-1-10 is used by nature. PMID:23724017

  1. Biochemical properties and atomic resolution structure of a proteolytically processed β-mannanase from cellulolytic Streptomyces sp. SirexAA-E.

    PubMed

    Takasuka, Taichi E; Acheson, Justin F; Bianchetti, Christopher M; Prom, Ben M; Bergeman, Lai F; Book, Adam J; Currie, Cameron R; Fox, Brian G

    2014-01-01

    β-Mannanase SACTE_2347 from cellulolytic Streptomyces sp. SirexAA-E is abundantly secreted into the culture medium during growth on cellulosic materials. The enzyme is composed of domains from the glycoside hydrolase family 5 (GH5), fibronectin type-III (Fn3), and carbohydrate binding module family 2 (CBM2). After secretion, the enzyme is proteolyzed into three different, catalytically active variants with masses of 53, 42 and 34 kDa corresponding to the intact protein, loss of the CBM2 domain, or loss of both the Fn3 and CBM2 domains. The three variants had identical N-termini starting with Ala51, and the positions of specific proteolytic reactions in the linker sequences separating the three domains were identified. To conduct biochemical and structural characterizations, the natural proteolytic variants were reproduced by cloning and heterologously expressed in Escherichia coli. Each SACTE_2347 variant hydrolyzed only β-1,4 mannosidic linkages, and also reacted with pure mannans containing partial galactosyl- and/or glucosyl substitutions. Examination of the X-ray crystal structure of the GH5 domain of SACTE_2347 suggests that two loops adjacent to the active site channel, which have differences in position and length relative to other closely related mannanases, play a role in producing the observed substrate selectivity. PMID:24710170

  2. M1 and M2 macrophage proteolytic and angiogenic profile analysis in atherosclerotic patients reveals a distinctive profile in type 2 diabetes.

    PubMed

    Roma-Lavisse, Charlotte; Tagzirt, Madjid; Zawadzki, Christophe; Lorenzi, Rodrigo; Vincentelli, André; Haulon, Stephan; Juthier, Francis; Rauch, Antoine; Corseaux, Delphine; Staels, Bart; Jude, Brigitte; Van Belle, Eric; Susen, Sophie; Chinetti-Gbaguidi, Giulia; Dupont, Annabelle

    2015-07-01

    This study aimed to investigate atherosclerotic mediators' expression levels in M1 and M2 macrophages and to focus on the influence of diabetes on M1/M2 profiles. Macrophages from 36 atherosclerotic patients (19 diabetics and 17 non-diabetics) were cultured with interleukin-1β (IL-1β) or IL-4 to induce M1 or M2 phenotype, respectively. The atherosclerotic mediators' expression was evaluated by quantitative reverse transcription-polymerase chain reaction (RT-PCR). The results showed that M1 and M2 macrophages differentially expressed mediators involved in proteolysis and angiogenesis processes. The proteolytic balance (matrix metalloproteinase-9 (MMP-9)/tissue inhibitor of metalloproteinase-1 (TIMP-1), MMP-9/plasminogen activator inhibitor-1 (PAI-1) and MMP-9/tissue factor pathway inhibitor-2 (TFPI-2) ratios) was higher in M1 versus M2, whereas M2 macrophages presented higher angiogenesis properties (increased vascular endothelial growth factor/TFPI-2 and tissue factor/TFPI-2 ratios). Moreover, M1 macrophages from diabetics displayed more important proangiogenic and proteolytic activities than non-diabetics. This study reveals that M1 and M2 macrophages could differentially modulate major atherosclerosis-related pathological processes. Moreover, M1 macrophages from diabetics display a deleterious phenotype that could explain the higher plaque vulnerability observed in these subjects. PMID:25966737

  3. The putative role of proteolytic pathways in the pathogenesis of Type 1 diabetes mellitus: the 'autophagy' hypothesis.

    PubMed

    Fierabracci, Alessandra

    2014-05-01

    Autoimmune diseases are a heterogeneous group of disorders affecting different organs and tissues. New tools, such as genome-wide association studies, have provided evidence for new susceptibility loci and candidate genes in the disease process including common susceptibility genes involved in the immunological synapse and T cell activation. Close linkages have been found in a number of diseases, including ankylosing spondylitis, multiple sclerosis, Crohn's disease and insulin-dependent diabetes mellitus (Type 1 diabetes mellitus). Evidence for some association with Type 1 diabetes was previously found in the region containing 5q15/ERAP1 (endoplasmic reticulum aminopeptidase 1) (rs30187, ARTS1). Recent data suggest that in eukaryotic cells in addition to the ubiquitin/proteasome system another proteolytic pathway may have a significant role in the autoimmunity process, i.e. the autophagic pathway which constitutes the principal regulated catabolic process mediated by lysosomes. Autophagy could play a role in MHC class I and class II self-antigen presentation at the basis of the autoimmunity process. Furthermore cross-talk among different proteolytic pathways was recently highlighted i.e. components processed in the ubiquitin/proteasome system possibly engaged in autophagic pathways. T1D is an autoimmune disease characterised by the destruction of pancreatic beta cells by autoreactive T cells. Immunological abnormalities can precede months to years the initial symptoms and clinical diagnosis. Our hypothesis suggests that in the autoimmune process autophagy can intervene at different levels, during the thymic selection process of T lymphocytes causing escape of autoreactive T cells, at the initiation stage of the disease, in the preclinical period or subsequently to the disease onset having a role at the level of perpetuation of the autoimmunity process. Supporting evidence derives from the already reported discovery of polymorphisms in autophagy-related genes in

  4. Proteolytic disassembly of peptide-mediated graphene oxide assemblies for turn-on fluorescence sensing of proteases

    NASA Astrophysics Data System (ADS)

    Yang, Jin-Kyoung; Kwak, Seon-Yeong; Jeon, Su-Ji; Lee, Eunjin; Ju, Jong-Min; Kim, Hye-In; Lee, Yoon-Sik; Kim, Jong-Ho

    2016-06-01

    Molecule-induced assembly of nanomaterials can alter their unique chemical and physical properties, which can be a promising approach for sensing. Herein, we demonstrate an optical `turn-on' biosensor for the detection of matrix metalloproteinase-2 (MMP-2), fabricated by means of a peptide-induced assembly of fluorescent graphene oxide (GO). Functionalization of GO with a peptide substrate for MMP-2 bearing a thiol group leads to its self-assembly via disulfide bonding, accompanied by self-quenching of GO's strong fluorescence. This peptide-induced GO assembly is then disassembled by proteolytic cleavage in the presence of MMP-2, thereby restoring the level of self-quenched GO fluorescence. With this approach, we are able to detect MMP-2 and to investigate the kinetic parameters of MMP-2 activity. The GO-peptide assembly is successfully applied to the selective and sensitive detection of MMP-2 secreted by living cells, human hepatocytes HepG2, at a concentration of 2 ng mL-1.Molecule-induced assembly of nanomaterials can alter their unique chemical and physical properties, which can be a promising approach for sensing. Herein, we demonstrate an optical `turn-on' biosensor for the detection of matrix metalloproteinase-2 (MMP-2), fabricated by means of a peptide-induced assembly of fluorescent graphene oxide (GO). Functionalization of GO with a peptide substrate for MMP-2 bearing a thiol group leads to its self-assembly via disulfide bonding, accompanied by self-quenching of GO's strong fluorescence. This peptide-induced GO assembly is then disassembled by proteolytic cleavage in the presence of MMP-2, thereby restoring the level of self-quenched GO fluorescence. With this approach, we are able to detect MMP-2 and to investigate the kinetic parameters of MMP-2 activity. The GO-peptide assembly is successfully applied to the selective and sensitive detection of MMP-2 secreted by living cells, human hepatocytes HepG2, at a concentration of 2 ng mL-1. Electronic

  5. Tetrahydrohyperforin Inhibits the Proteolytic Processing of Amyloid Precursor Protein and Enhances Its Degradation by Atg5-Dependent Autophagy

    PubMed Central

    Muñoz, Vanessa C.; Yefi, Claudia P.; Bustamante, Hianara A.; Barraza, Rafael R.; Tapia-Rojas, Cheril; Otth, Carola; Barrera, María José; González, Carlos; Mardones, Gonzalo A.; Inestrosa, Nibaldo C.; Burgos, Patricia V.

    2015-01-01

    Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid-β (Aβ) peptide. We have previously shown that the compound tetrahydrohyperforin (IDN5706) prevents accumulation of Aβ species in an in vivo model of AD, however the mechanism that explains this reduction is not well understood. We show herein that IDN5706 decreases the levels of ER degradation enhancer, mannosidase alpha-like 1 (EDEM1), a key chaperone related to endoplasmic-reticulum-associated degradation (ERAD). Moreover, we observed that low levels of EDEM1 correlated with a strong activation of autophagy, suggesting a crosstalk between these two pathways. We observed that IDN5706 perturbs the glycosylation and proteolytic processing of the amyloid precursor protein (APP), resulting in the accumulation of immature APP (iAPP) in the endoplasmic reticulum. To investigate the contribution of autophagy, we tested the effect of IDN5706 in Atg5-depleted cells. We found that depletion of Atg5 enhanced the accumulation of iAPP in response to IDN5706 by slowing down its degradation. Our findings reveal that IDN5706 promotes degradation of iAPP via the activation of Atg5-dependent autophagy, shedding light on the mechanism that may contribute to the reduction of Aβ production in vivo. PMID:26308941

  6. Tetrahydrohyperforin Inhibits the Proteolytic Processing of Amyloid Precursor Protein and Enhances Its Degradation by Atg5-Dependent Autophagy.

    PubMed

    Cavieres, Viviana A; González, Alexis; Muñoz, Vanessa C; Yefi, Claudia P; Bustamante, Hianara A; Barraza, Rafael R; Tapia-Rojas, Cheril; Otth, Carola; Barrera, María José; González, Carlos; Mardones, Gonzalo A; Inestrosa, Nibaldo C; Burgos, Patricia V

    2015-01-01

    Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid-β (Aβ) peptide. We have previously shown that the compound tetrahydrohyperforin (IDN5706) prevents accumulation of Aβ species in an in vivo model of AD, however the mechanism that explains this reduction is not well understood. We show herein that IDN5706 decreases the levels of ER degradation enhancer, mannosidase alpha-like 1 (EDEM1), a key chaperone related to endoplasmic-reticulum-associated degradation (ERAD). Moreover, we observed that low levels of EDEM1 correlated with a strong activation of autophagy, suggesting a crosstalk between these two pathways. We observed that IDN5706 perturbs the glycosylation and proteolytic processing of the amyloid precursor protein (APP), resulting in the accumulation of immature APP (iAPP) in the endoplasmic reticulum. To investigate the contribution of autophagy, we tested the effect of IDN5706 in Atg5-depleted cells. We found that depletion of Atg5 enhanced the accumulation of iAPP in response to IDN5706 by slowing down its degradation. Our findings reveal that IDN5706 promotes degradation of iAPP via the activation of Atg5-dependent autophagy, shedding light on the mechanism that may contribute to the reduction of Aβ production in vivo. PMID:26308941

  7. Schistosoma mansoni: possible involvement of protein kinase C in linoleic acid-induced proteolytic enzyme release from cercariae.

    PubMed

    Matsumura, K; Mitsui, Y; Sato, K; Sakamoto, M; Aoki, Y

    1991-04-01

    The possible involvement of protein kinase C and Ca2+ metabolism in the proteolytic enzyme release from schistosome cercariae was studied. Cercariae were placed in dechlorinated tap water containing 0.37 mM calcium in the small glass petri dish and exposed to the stimuli (linoleic acid, phorbol esters, and Ca2+ ionophore) with or without inhibitors of protein kinase C or Ca2+ metabolism. The proteolytic activity of incubation medium of cercariae thus treated was measured by the azocoll assay. The penetration response of cercariae induced by linoleic acid, a physiological stimulus, was mimicked by phorbol esters. When exposed to phorbol esters, 0.02 to 2 microM of 12-O-tetradecanoylphorbol-13-acetate (TPA) and 0.2 to 2 microM of phorbol-12,13-dibutyrate (PDBu), cercariae ceased the swimming movement, began a rhythmic thrusting of the anterior tip of the parasite, and released the proteolytic enzyme, but they did not shed the tails. Lowering Ca2+ in water by addition of 5 mM ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), phorbol ester-induced release of enzyme was completely inhibited. Phorbol ester-induced release of enzyme was partially inhibited by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an inhibitor of protein kinase C, at a concentration of 100 microM. H-7 alone, at a concentration of 100 microM, did not affect the swimming movement of cercariae. The cercariae were stimulated to release the enzyme by high concentrations (10 and 100 microM) of the Ca2+ ionophore, A23187, but enzyme was not released by low concentrations (0.5 and 1 microM) of this drug. Cercariae exposed to A23187 behaved differently from those exposed to phorbol esters. They ceased swimming, showed strong muscle contraction, and shed their tail. A23187 stimulated cercariae to release the enzyme in the water containing 5 mM EGTA. A23187-induced enzyme release was not inhibited by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin

  8. Epitope Structure of the Carbohydrate Recognition Domain of Asialoglycoprotein Receptor to a Monoclonal Antibody Revealed by High-Resolution Proteolytic Excision Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Stefanescu, Raluca; Born, Rita; Moise, Adrian; Ernst, Beat; Przybylski, Michael

    2011-01-01

    Recent studies suggest that the H1 subunit of the carbohydrate recognition domain (H1CRD) of the asialoglycoprotein receptor is used as an entry site into hepatocytes by hepatitis A and B viruses and Marburg virus. Thus, molecules binding specifically to the CRD might exert inhibition towards these diseases by blocking the virus entry site. We report here the identification of the epitope structure of H1CRD to a monoclonal antibody by proteolytic epitope excision of the immune complex and high-resolution MALDI-FTICR mass spectrometry. As a prerequisite of the epitope determination, the primary structure of the H1CRD antigen was characterised by ESI-FTICR-MS of the intact protein and by LC-MS/MS of tryptic digest mixtures. Molecular mass determination and proteolytic fragments provided the identification of two intramolecular disulfide bridges (seven Cys residues), and a Cys-mercaptoethanol adduct formed by treatment with β-mercaptoethanol during protein extraction. The H1CRD antigen binds to the monoclonal antibody in both native and Cys-alkylated form. For identification of the epitope, the antibody was immobilized on N-hydroxysuccinimide (NHS)-activated Sepharose. Epitope excision and epitope extraction with trypsin and FTICR-MS of affinity-bound peptides provided the identification of two specific epitope peptides (5-16) and (17-23) that showed high affinity to the antibody. Affinity studies of the synthetic epitope peptides revealed independent binding of each peptide to the antibody.

  9. Anti-Inflammatory Benefits of Antibiotic-Induced Neutrophil Apoptosis: Tulathromycin Induces Caspase-3-Dependent Neutrophil Programmed Cell Death and Inhibits NF-κB Signaling and CXCL8 Transcription▿

    PubMed Central

    Fischer, Carrie D.; Beatty, Jennifer K.; Zvaigzne, Cheryl G.; Morck, Douglas W.; Lucas, Merlyn J.; Buret, A. G.

    2011-01-01

    Clearance of apoptotic neutrophils is a central feature of the resolution of inflammation. Findings indicate that immuno-modulation and induction of neutrophil apoptosis by macrolide antibiotics generate anti-inflammatory benefits via mechanisms that remain obscure. Tulathromycin (TUL), a new antimicrobial agent for bovine respiratory disease, offers superior clinical efficacy for reasons not fully understood. The aim of this study was to identify the immuno-modulating effects of tulathromycin and, in this process, to establish tulathromycin as a new model for characterizing the novel anti-inflammatory properties of antibiotics. Bronchoalveolar lavage specimens were collected from Holstein calves 3 and 24 h postinfection, challenged intratracheally with live Mannheimia haemolytica (2 × 107 CFU), and treated with vehicle or tulathromycin (2.5 mg/kg body weight). Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining and enzyme-linked immunosorbent assay (ELISA) revealed that tulathromycin treatment significantly increased leukocyte apoptosis and reduced levels of proinflammatory leukotriene B4 in M. haemolytica-challenged calves. In vitro, tulathromycin concentration dependently induced apoptosis in freshly isolated bovine neutrophils from healthy steers in a capase-3-dependent manner but failed to induce apoptosis in bovine fibroblasts, epithelial cells, and endothelial cells, as well as freshly isolated bovine blood monocytes and monocyte-derived macrophages. The proapoptotic effects of TUL were also, in part, drug specific; equimolar concentrations of penicillin G, oxytetracycline, and ceftiofur failed to cause apoptosis in bovine neutrophils. In addition, tulathromycin significantly reduced levels of phosphorylated IκBα, nuclear translocation of NF-κB p65, and mRNA levels of proinflammatory interleukin-8 in lipopolysaccharide (LPS)-stimulated bovine neutrophils. The findings illustrate novel mechanisms through which tulathromycin confers anti-inflammatory benefits. PMID:20956586

  10. A novel regulation of PD-1 ligands on mesenchymal stromal cells through MMP-mediated proteolytic cleavage

    PubMed Central

    Dezutter-Dambuyant, Colette; Durand, Isabelle; Alberti, Laurent; Bendriss-Vermare, Nathalie; Valladeau-Guilemond, Jenny; Duc, Adeline; Magron, Audrey; Morel, Anne-Pierre; Sisirak, Vanja; Rodriguez, Céline; Cox, David; Olive, Daniel; Caux, Christophe

    2016-01-01

    ABSTRACT Whether fibroblasts regulate immune response is a crucial issue in the modulation of inflammatory responses. Herein, we demonstrate that foreskin fibroblasts (FFs) potently inhibit CD3+ T cell proliferation through a mechanism involving early apoptosis of activated T cells. Using blocking antibodies, we demonstrate that the inhibition of T cell proliferation occurs through cell-to-cell interactions implicating PD-1 receptor expressed on T cells and its ligands, PD-L1 and PD-L2, on fibroblasts. Dual PD-1 ligand neutralization is required to abrogate (i) binding of the PD-1-Fc fusion protein, (ii) early apoptosis of T cells, and (iii) inhibition of T cell proliferation. Of utmost importance, we provide the first evidence that PD-1 ligand expression is regulated through proteolytic cleavage by endogenous matrix metalloproteinases (MMPs) without transcriptional alteration during culture-time. Using (i) different purified enzymatic activities, (ii) MMP-specific inhibitors, and (iii) recombinant human MMP-9 and MMP-13, we demonstrated that in contrast to CD80/CD86, PD-L1 was selectively cleaved by MMP-13, while PD-L2 was sensitive to broader MMP activities. Their cleavage by exogenous MMP-9 and MMP-13 with loss of PD-1 binding domain resulted in the reversion of apoptotic signals on mitogen-activated CD3+ T cells. We suggest that MMP-dependent cleavage of PD-1 ligands on fibroblasts may limit their immunosuppressive capacity and thus contribute to the exacerbation of inflammation in tissues. In contrast, carcinoma-associated fibroblasts appear PD-1 ligand-depleted through MMP activity that may impair physical deletion of exhausted defective memory T cells through apoptosis and facilitate their regulatory functions. These observations should be considered when using the powerful PD-1/PD-L1 blocking immunotherapies. PMID:27141350

  11. Proteolytic disassembly of peptide-mediated graphene oxide assemblies for turn-on fluorescence sensing of proteases.

    PubMed

    Yang, Jin-Kyoung; Kwak, Seon-Yeong; Jeon, Su-Ji; Lee, Eunjin; Ju, Jong-Min; Kim, Hye-In; Lee, Yoon-Sik; Kim, Jong-Ho

    2016-06-16

    Molecule-induced assembly of nanomaterials can alter their unique chemical and physical properties, which can be a promising approach for sensing. Herein, we demonstrate an optical 'turn-on' biosensor for the detection of matrix metalloproteinase-2 (MMP-2), fabricated by means of a peptide-induced assembly of fluorescent graphene oxide (GO). Functionalization of GO with a peptide substrate for MMP-2 bearing a thiol group leads to its self-assembly via disulfide bonding, accompanied by self-quenching of GO's strong fluorescence. This peptide-induced GO assembly is then disassembled by proteolytic cleavage in the presence of MMP-2, thereby restoring the level of self-quenched GO fluorescence. With this approach, we are able to detect MMP-2 and to investigate the kinetic parameters of MMP-2 activity. The GO-peptide assembly is successfully applied to the selective and sensitive detection of MMP-2 secreted by living cells, human hepatocytes HepG2, at a concentration of 2 ng mL(-1). PMID:27271225

  12. The role of proteolytic processing and the stable signal peptide in expression of the Old World arenavirus envelope glycoprotein ectodomain

    SciTech Connect

    Burri, Dominique J.; Pasquato, Antonella; Ramos da Palma, Joel; Igonet, Sebastien; Oldstone, Michael B.A.; Kunz, Stefan

    2013-02-05

    Maturation of the arenavirus GP precursor (GPC) involves proteolytic processing by cellular signal peptidase and the proprotein convertase subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P), yielding a tripartite complex comprised of a stable signal peptide (SSP), the receptor-binding GP1, and the fusion-active transmembrane GP2. Here we investigated the roles of SKI-1/S1P processing and SSP in the biosynthesis of the recombinant GP ectodomains of lymphocytic choriomeningitis virus (LCMV) and Lassa virus (LASV). When expressed in mammalian cells, the LCMV and LASV GP ectodomains underwent processing by SKI-1/S1P, followed by dissociation of GP1 from GP2. The GP2 ectodomain spontaneously formed trimers as revealed by chemical cross-linking. The endogenous SSP, known to be crucial for maturation and transport of full-length arenavirus GPC was dispensable for processing and secretion of the soluble GP ectodomain, suggesting a specific role of SSP in the stable prefusion conformation and transport of full-length GPC.

  13. A novel proteasome inhibitor suppresses tumor growth via targeting both 19S proteasome deubiquitinases and 20S proteolytic peptidases

    PubMed Central

    Liu, Ningning; Liu, Chunjiao; Li, Xiaofen; Liao, Siyan; Song, Wenbin; Yang, Changshan; Zhao, Chong; Huang, Hongbiao; Guan, Lixia; Zhang, Peiquan; Liu, Shouting; Hua, Xianliang; Chen, Xin; Zhou, Ping; Lan, Xiaoying; Yi, Songgang; Wang, Shunqing; Wang, Xuejun; Dou, Q. Ping; Liu, Jinbao

    2014-01-01

    The successful development of bortezomib-based therapy for treatment of multiple myeloma has established proteasome inhibition as an effective therapeutic strategy, and both 20S proteasome peptidases and 19S deubiquitinases (DUBs) are becoming attractive targets of cancer therapy. It has been reported that metal complexes, such as copper complexes, inhibit tumor proteasome. However, the involved mechanism of action has not been fully characterized. Here we report that (i) copper pyrithione (CuPT), an alternative to tributyltin for antifouling paint biocides, inhibits the ubiquitin-proteasome system (UPS) via targeting both 19S proteasome-specific DUBs and 20S proteolytic peptidases with a mechanism distinct from that of the FDA-approved proteasome inhibitor bortezomib; (ii) CuPT potently inhibits proteasome-specific UCHL5 and USP14 activities; (iii) CuPT inhibits tumor growth in vivo and induces cytotoxicity in vitro and ex vivo. This study uncovers a novel class of dual inhibitors of DUBs and proteasome and suggests a potential clinical strategy for cancer therapy. PMID:24912524

  14. Sequence motif upstream of the Hendra virus fusion protein cleavage site is not sufficient to promote efficient proteolytic processing

    SciTech Connect

    Craft, Willie Warren; Dutch, Rebecca Ellis . E-mail: rdutc2@uky.edu

    2005-10-10

    The Hendra virus fusion (HeV F) protein is synthesized as a precursor, F{sub 0}, and proteolytically cleaved into the mature F{sub 1} and F{sub 2} heterodimer, following an HDLVDGVK{sub 109} motif. This cleavage event is required for fusogenic activity. To determine the amino acid requirements for processing of the HeV F protein, we constructed multiple mutants. Individual and simultaneous alanine substitutions of the eight residues immediately upstream of the cleavage site did not eliminate processing. A chimeric SV5 F protein in which the furin site was substituted for the VDGVK{sub 109} motif of the HeV F protein was not processed but was expressed on the cell surface. Another chimeric SV5 F protein containing the HDLVDGVK{sub 109} motif of the HeV F protein underwent partial cleavage. These data indicate that the upstream region can play a role in protease recognition, but is neither absolutely required nor sufficient for efficient processing of the HeV F protein.

  15. A novel proteasome inhibitor suppresses tumor growth via targeting both 19S proteasome deubiquitinases and 20S proteolytic peptidases.

    PubMed

    Liu, Ningning; Liu, Chunjiao; Li, Xiaofen; Liao, Siyan; Song, Wenbin; Yang, Changshan; Zhao, Chong; Huang, Hongbiao; Guan, Lixia; Zhang, Peiquan; Liu, Shouting; Hua, Xianliang; Chen, Xin; Zhou, Ping; Lan, Xiaoying; Yi, Songgang; Wang, Shunqing; Wang, Xuejun; Dou, Q Ping; Liu, Jinbao

    2014-01-01

    The successful development of bortezomib-based therapy for treatment of multiple myeloma has established proteasome inhibition as an effective therapeutic strategy, and both 20S proteasome peptidases and 19S deubiquitinases (DUBs) are becoming attractive targets of cancer therapy. It has been reported that metal complexes, such as copper complexes, inhibit tumor proteasome. However, the involved mechanism of action has not been fully characterized. Here we report that (i) copper pyrithione (CuPT), an alternative to tributyltin for antifouling paint biocides, inhibits the ubiquitin-proteasome system (UPS) via targeting both 19S proteasome-specific DUBs and 20S proteolytic peptidases with a mechanism distinct from that of the FDA-approved proteasome inhibitor bortezomib; (ii) CuPT potently inhibits proteasome-specific UCHL5 and USP14 activities; (iii) CuPT inhibits tumor growth in vivo and induces cytotoxicity in vitro and ex vivo. This study uncovers a novel class of dual inhibitors of DUBs and proteasome and suggests a potential clinical strategy for cancer therapy. PMID:24912524

  16. The role of proteolytic processing and the stable signal peptide in expression of the Old World arenavirus envelope glycoprotein ectodomain

    PubMed Central

    Burri, Dominique J.; Pasquato, Antonella; da Palma, Joel Ramos; Igonet, Sebastien; Oldstone, Michael B.A.; Kunz, Stefan

    2012-01-01

    Maturation of the arenavirus GP precursor (GPC) involves proteolytic processing by cellular signal peptidase and the proprotein convertase subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P), yielding a tripartite complex comprised of a stable signal peptide (SSP), the receptor-binding GP1, and the fusion-active transmembrane GP2. Here we investigated the roles of SKI-1/S1P processing and SSP in the biosynthesis of the recombinant GP ectodomains of lymphocytic choriomeningitis virus (LCMV) and Lassa virus (LASV). When expressed in mammalian cells, the LCMV and LASV GP ectodomains underwent processing by SKI-1/S1P, followed by dissociation of GP1 from GP2. The GP2 ectodomain spontaneously formed trimers as revealed by chemical cross-linking. The endogenous SSP, known to be crucial for maturation and transport of full-length arenavirus GPC was dispensable for processing and secretion of the soluble GP ectodomain, suggesting a specific role of SSP in the stable prefusion conformation and transport of full-length GPC. PMID:23218200

  17. Sensitive microplate assay for the detection of proteolytic enzymes using radiolabeled gelatin

    SciTech Connect

    Robertson, B.D.; Kwan-Lim, G.E.; Maizels, R.M.

    1988-07-01

    A sensitive, microplate assay is described for the detection of a wide range of proteolytic enzymes, using radio-iodine-labeled gelatin as substrate. The technique uses the Bolton-Hunter reagent to label the substrate, which is then coated onto the wells of polyvinyl chloride microtiter plates. By measuring the radioactivity released the assay is able to detect elastase, trypsin, and collagenase in concentrations of 1 ng/ml or less, while the microtiter format permits multiple sample handling and minimizes sample volumes required for analysis.

  18. Colon tumour secretopeptidome: insights into endogenous proteolytic cleavage events in the colon tumour microenvironment.

    PubMed

    Greening, David W; Kapp, Eugene A; Ji, Hong; Speed, Terry P; Simpson, Richard J

    2013-11-01

    The secretopeptidome comprises endogenous peptides derived from proteins secreted into the tumour microenvironment through classical and non-classical secretion. This study characterised the low-Mr (<3kDa) component of the human colon tumour (LIM1215, LIM1863) secretopeptidome, as a first step towards gaining insights into extracellular proteolytic cleavage events in the tumour microenvironment. Based on two biological replicates, this secretopeptidome isolation strategy utilised differential centrifugal ultrafiltration in combination with analytical RP-HPLC and nanoLC-MS/MS. Secreted peptides were identified using a combination of Mascot and post-processing analyses including MSPro re-scoring, extended feature sets and Percolator, resulting in 474 protein identifications from 1228 peptides (≤1% q-value, ≤5% PEP) - a 36% increase in peptide identifications when compared with conventional Mascot (homology ionscore thresholding). In both colon tumour models, 122 identified peptides were derived from 41 cell surface protein ectodomains, 23 peptides (12 proteins) from regulated intramembrane proteolysis (RIP), and 12 peptides (9 proteins) generated from intracellular domain proteolysis. Further analyses using the protease/substrate database MEROPS, (http://merops.sanger.ac.uk/), revealed 335 (71%) proteins classified as originating from classical/non-classical secretion, or the cell membrane. Of these, peptides were identified from 42 substrates in MEROPS with defined protease cleavage sites, while peptides generated from a further 205 substrates were fragmented by hitherto unknown proteases. A salient finding was the identification of peptides from 88 classical/non-classical secreted substrates in MEROPS, implicated in tumour progression and angiogenesis (FGFBP1, PLXDC2), cell-cell recognition and signalling (DDR1, GPA33), and tumour invasiveness and metastasis (MACC1, SMAGP); the nature of the proteases responsible for these proteolytic events is unknown. To

  19. Histone H3.3 and its proteolytically processed form drive a cellular senescence program

    PubMed Central

    Duarte, Luis F.; Young, Andrew R. J.; Wang, Zichen; Wu, Hsan-Au; Panda, Taniya; Kou, Yan; Kapoor, Avnish; Hasson, Dan; Mills, Nicholas R.; Ma’ayan, Avi; Narita, Masashi; Bernstein, Emily

    2014-01-01

    The process of cellular senescence generates a repressive chromatin environment, however, the role of histone variants and histone proteolytic cleavage in senescence remains unclear. Using models of oncogene-induced and replicative senescence, here we report novel histone H3 tail cleavage events mediated by the protease Cathepsin L. We find that cleaved forms of H3 are nucleosomal and the histone variant H3.3 is the preferred cleaved form of H3. Ectopic expression of H3.3 and its cleavage product (H3.3cs1), which lacks the first twenty-one amino acids of the H3 tail, is sufficient to induce senescence. Further, H3.3cs1 chromatin incorporation is mediated by the HUCA histone chaperone complex. Genome-wide transcriptional profiling revealed that H3.3cs1 facilitates transcriptional silencing of cell cycle regulators including RB/E2F target genes, likely via the permanent removal of H3K4me3. Collectively, our study identifies histone H3.3 and its proteolytically processed forms as key regulators of cellular senescence. PMID:25394905

  20. CleavPredict: A Platform for Reasoning about Matrix Metalloproteinases Proteolytic Events

    PubMed Central

    Kumar, Sonu; Ratnikov, Boris I.; Kazanov, Marat D.; Smith, Jeffrey W.; Cieplak, Piotr

    2015-01-01

    CleavPredict (http://cleavpredict.sanfordburnham.org) is a Web server for substrate cleavage prediction for matrix metalloproteinases (MMPs). It is intended as a computational platform aiding the scientific community in reasoning about proteolytic events. CleavPredict offers in silico prediction of cleavage sites specific for 11 human MMPs. The prediction method employs the MMP specific position weight matrices (PWMs) derived from statistical analysis of high-throughput phage display experimental results. To augment the substrate cleavage prediction process, CleavPredict provides information about the structural features of potential cleavage sites that influence proteolysis. These include: secondary structure, disordered regions, transmembrane domains, and solvent accessibility. The server also provides information about subcellular location, co-localization, and co-expression of proteinase and potential substrates, along with experimentally determined positions of single nucleotide polymorphism (SNP), and posttranslational modification (PTM) sites in substrates. All this information will provide the user with perspectives in reasoning about proteolytic events. CleavPredict is freely accessible, and there is no login required. PMID:25996941

  1. CleavPredict: A Platform for Reasoning about Matrix Metalloproteinases Proteolytic Events.

    PubMed

    Kumar, Sonu; Ratnikov, Boris I; Kazanov, Marat D; Smith, Jeffrey W; Cieplak, Piotr

    2015-01-01

    CleavPredict (http://cleavpredict.sanfordburnham.org) is a Web server for substrate cleavage prediction for matrix metalloproteinases (MMPs). It is intended as a computational platform aiding the scientific community in reasoning about proteolytic events. CleavPredict offers in silico prediction of cleavage sites specific for 11 human MMPs. The prediction method employs the MMP specific position weight matrices (PWMs) derived from statistical analysis of high-throughput phage display experimental results. To augment the substrate cleavage prediction process, CleavPredict provides information about the structural features of potential cleavage sites that influence proteolysis. These include: secondary structure, disordered regions, transmembrane domains, and solvent accessibility. The server also provides information about subcellular location, co-localization, and co-expression of proteinase and potential substrates, along with experimentally determined positions of single nucleotide polymorphism (SNP), and posttranslational modification (PTM) sites in substrates. All this information will provide the user with perspectives in reasoning about proteolytic events. CleavPredict is freely accessible, and there is no login required. PMID:25996941

  2. Endosomal localization of TLR8 confers distinctive proteolytic processing on human myeloid cells.

    PubMed

    Ishii, Noriko; Funami, Kenji; Tatematsu, Megumi; Seya, Tsukasa; Matsumoto, Misako

    2014-11-15

    Nucleic acid-sensing TLRs are involved in both antimicrobial immune responses and autoimmune inflammation. TLR8 is phylogenetically and structurally related to TLR7 and TLR9, which undergo proteolytic processing in the endolysosomes to generate functional receptors. Recent structural analyses of human TLR8 ectodomain and its liganded form demonstrated that TLR8 is also cleaved, and both the N- and C-terminal halves contribute to ligand binding. However, the structures and ssRNA recognition mode of endogenous TLR8 in human primary cells are largely unknown. In this study, we show that proteolytic processing of TLR8 occurs in human monocytes and macrophages in a different manner compared with TLR7/9 cleavage. The insertion loop between leucine-rich repeats 14 and 15 in TLR8 is indispensable for the cleavage and stepwise processing that occurs in the N-terminal fragment. Both furin-like proprotein convertase and cathepsins contribute to TLR8 cleavage in the early/late endosomes. TLR8 recognizes viral ssRNA and endogenous RNA, such as microRNAs, resulting in the production of proinflammatory cytokines. Hence, localization sites of the receptors are crucial for the nucleic acid-sensing mode and downstream signaling. PMID:25297876

  3. Proteolytic processing of Alzheimer’s β-amyloid precursor protein

    PubMed Central

    Zhang, Han; Ma, Qilin; Zhang, Yun-wu; Xu, Huaxi

    2011-01-01

    β–amyloid precursor protein (APP) is a critical factor in the pathogenesis of Alzheimer’s disease (AD). APP undergoes posttranslational proteolysis/processing to generate the hydrophobic β-amyloid (Aβ) peptides. Deposition of Aβ in the brain, forming oligomeric Aβ and plaques, is identified as one of the key pathological hallmarks of AD. The processing of APP to generate Aβ is executed by β- and γ-secretase and is highly regulated. Aβ toxicity can lead to synaptic dysfunction, neuronal cell death, impaired learning/memory and abnormal behaviors in AD models in vitro and in vivo. Aside from Aβ, proteolytic cleavages of APP can also give rise to the APP intracellular domain (AICD), reportedly involved in multiple types of cellular events such as gene transcription and apoptotic cell death. In addition to amyloidogenic processing, APP can also be cleaved by α-secretase to form a soluble or secreted APP ectodomain (sAPP-α) that has been shown to be mostly neuro-protective. In this review, we describe the mechanisms involved in APP metabolism and the likely functions of its various proteolytic products to give a better understanding of the patho/physiological functions of APP. PMID:22122372

  4. Intrinsic Tau Acetylation Is Coupled to Auto-Proteolytic Tau Fragmentation

    PubMed Central

    Cohen, Todd J.; Constance, Brian H.; Hwang, Andrew W.; James, Michael; Yuan, Chao-Xing

    2016-01-01

    Tau proteins are abnormally aggregated in a range of neurodegenerative tauopathies including Alzheimer’s disease (AD). Recently, tau has emerged as an extensively post-translationally modified protein, among which lysine acetylation is critical for normal tau function and its pathological aggregation. Here, we demonstrate that tau isoforms have different propensities to undergo lysine acetylation, with auto-acetylation occurring more prominently within the lysine-rich microtubule-binding repeats. Unexpectedly, we identified a unique intrinsic property of tau in which auto-acetylation induces proteolytic tau cleavage, thereby generating distinct N- and C-terminal tau fragments. Supporting a catalytic reaction-based mechanism, mapping and mutagenesis studies showed that tau cysteines, which are required for acetyl group transfer, are also essential for auto-proteolytic tau processing. Further mass spectrometry analysis identified the C-terminal 2nd and 4th microtubule binding repeats as potential sites of auto-cleavage. The identification of acetylation-mediated auto-proteolysis provides a new biochemical mechanism for tau self-regulation and warrants further investigation into whether auto-catalytic functions of tau are implicated in AD and other tauopathies. PMID:27383765

  5. Electrochemical sensing system employing fructosamine 6-kinase enables glycated albumin measurement requiring no proteolytic digestion.

    PubMed

    Kameya, Miho; Tsugawa, Wakako; Yamada-Tajima, Mayumi; Hatada, Mika; Suzuki, Keita; Sakaguchi-Mikami, Akane; Ferri, Stefano; Klonoff, David C; Sode, Koji

    2016-06-01

    Currently available enzymatic methods for the measurement of glycated proteins utilize fructosyl amino acid/peptide oxidases (FAOXs/FPOXs) as sensing elements. FAOXs/FPOXs oxidize glycated amino acids or glycated dipeptides but they are not able to accept longer glycated peptides or intact glycated proteins as substrates. Therefore, pretreatment via proteolytic digestion is unavoidable with the current enzymatic methods, and there remains a need for simpler measurement methods for glycated proteins. In this study, in order to develop a novel sensing system for glycated albumin (GA), a marker for diabetes, with no requirement for proteolytic digestion, we created an electrochemical sensor based on fructosamine 6-kinase (FN6K) from Escherichia coli. Uniquely, FN6K can react directly with intact GA unlike FAOXs/FPOXs. The concentration of GA in samples was measured using a carbon-printed disposable electrode upon which FN6K as well as two additional enzymes, pyruvate kinase and pyruvate dehydrogenase were overlaid. A clear correlation between the response current and the concentration of GA was observed in the range of 20-100 µM GA, which is suitable for measurement of GA in diluted blood samples from both healthy individuals and patients with diabetes. The sensing system reported here could be applied to point-of-care-testing devices for measurement of glycated proteins. PMID:27067959

  6. Preliminary investigation of intrinsic UV fluorescence spectroscopic changes associated with proteolytic digestion of bovine articular cartilage

    NASA Astrophysics Data System (ADS)

    Lewis, William; Padilla-Martinez, Juan-Pablo; Ortega-Martinez, Antonio; Franco, Walfre

    2016-03-01

    Degradation and destruction of articular cartilage is the etiology of osteoarthritis (OA), an entity second only to cardiovascular disease as a cause of disability in the United States. Joint mechanics and cartilage biochemistry are believed to play a role in OA; an optical tool to detect structural and chemical changes in articular cartilage might offer benefit for its early detection and treatment. The objective of the present study was to identify the spectral changes in intrinsic ultraviolet (UV) fluorescence of cartilage that occur after proteolytic digestion of cartilage. Bovine articular cartilage samples were incubated in varying concentrations of collagenase ranging from 10ug/mL up to 5mg/mL for 18 hours at 37°C, a model of OA. Pre- and post-incubation measurements were taken of the UV excitation-emission spectrum of each cartilage sample. Mechanical tests were performed to determine the pre- and post-digestion force/displacement ratio associated with indentation of each sample. Spectral changes in intrinsic cartilage fluorescence and stiffness of the cartilage were associated with proteolytic digestion. In particular, changes in the relative intensity of fluorescence peaks associated with pentosidine crosslinks (330 nm excitation, 390 nm emission) and tryptophan (290 nm excitation, 340 nm emission) were found to correlate with different degrees of cartilage digestion and cartilage stiffness. In principle, it may be possible to use UV fluorescence spectral data for early detection of damage to articular cartilage, and as a surrogate measure for cartilage stiffness.

  7. Inflammatory Proprotein Convertase-Matrix Metalloproteinase Proteolytic Pathway in Antigen-presenting Cells as a Step to Autoimmune Multiple Sclerosis*

    PubMed Central

    Shiryaev, Sergey A.; Remacle, Albert G.; Savinov, Alexei Y.; Chernov, Andrei V.; Cieplak, Piotr; Radichev, Ilian A.; Williams, Roy; Shiryaeva, Tatiana N.; Gawlik, Katarzyna; Postnova, Tatiana I.; Ratnikov, Boris I.; Eroshkin, Alexei M.; Motamedchaboki, Khatereh; Smith, Jeffrey W.; Strongin, Alex Y.

    2009-01-01

    Multiple sclerosis (MS) is a disease of the central nervous system with autoimmune etiology. Susceptibility to MS is linked to viral and bacterial infections. Matrix metalloproteinases (MMPs) play a significant role in the fragmentation of myelin basic protein (MBP) and demyelination. The splice variants of the single MBP gene are expressed in the oligodendrocytes of the central nervous system (classic MBP) and in the immune cells (Golli-MBPs). Our data suggest that persistent inflammation caused by environmental risk factors is a step to MS. We have discovered biochemical evidence suggesting the presence of the inflammatory proteolytic pathway leading to MS. The pathway involves the self-activated furin and PC2 proprotein convertases and membrane type-6 MMP (MT6-MMP/MMP-25) that is activated by furin/PC2. These events are followed by MMP-25 proteolysis of the Golli-MBP isoforms in the immune system cells and stimulation of the specific autoimmune T cell clones. It is likely that the passage of these autoimmune T cell clones through the disrupted blood-brain barrier to the brain and the recognition of neuronal, classic MBP causes inflammation leading to the further up-regulation of the activity of the multiple individual MMPs, the massive cleavage of MBP in the brain, demyelination, and MS. In addition to the cleavage of Golli-MBPs, MMP-25 proteolysis readily inactivates crystallin αB that is a suppressor of MS. These data suggest that MMP-25 plays an important role in MS pathology and that MMP-25, especially because of its restricted cell/tissue expression pattern and cell surface/lipid raft localization, is a promising drug target in MS. PMID:19726693

  8. Inflammatory proprotein convertase-matrix metalloproteinase proteolytic pathway in antigen-presenting cells as a step to autoimmune multiple sclerosis.

    PubMed

    Shiryaev, Sergey A; Remacle, Albert G; Savinov, Alexei Y; Chernov, Andrei V; Cieplak, Piotr; Radichev, Ilian A; Williams, Roy; Shiryaeva, Tatiana N; Gawlik, Katarzyna; Postnova, Tatiana I; Ratnikov, Boris I; Eroshkin, Alexei M; Motamedchaboki, Khatereh; Smith, Jeffrey W; Strongin, Alex Y

    2009-10-30

    Multiple sclerosis (MS) is a disease of the central nervous system with autoimmune etiology. Susceptibility to MS is linked to viral and bacterial infections. Matrix metalloproteinases (MMPs) play a significant role in the fragmentation of myelin basic protein (MBP) and demyelination. The splice variants of the single MBP gene are expressed in the oligodendrocytes of the central nervous system (classic MBP) and in the immune cells (Golli-MBPs). Our data suggest that persistent inflammation caused by environmental risk factors is a step to MS. We have discovered biochemical evidence suggesting the presence of the inflammatory proteolytic pathway leading to MS. The pathway involves the self-activated furin and PC2 proprotein convertases and membrane type-6 MMP (MT6-MMP/MMP-25) that is activated by furin/PC2. These events are followed by MMP-25 proteolysis of the Golli-MBP isoforms in the immune system cells and stimulation of the specific autoimmune T cell clones. It is likely that the passage of these autoimmune T cell clones through the disrupted blood-brain barrier to the brain and the recognition of neuronal, classic MBP causes inflammation leading to the further up-regulation of the activity of the multiple individual MMPs, the massive cleavage of MBP in the brain, demyelination, and MS. In addition to the cleavage of Golli-MBPs, MMP-25 proteolysis readily inactivates crystallin alphaB that is a suppressor of MS. These data suggest that MMP-25 plays an important role in MS pathology and that MMP-25, especially because of its restricted cell/tissue expression pattern and cell surface/lipid raft localization, is a promising drug target in MS. PMID:19726693

  9. Evidence that the glucoamylases and alpha-amylase secreted by Aspergillus niger are proteolytically processed products of a precursor enzyme.

    PubMed

    Dubey, A K; Suresh, C; Kavitha, R; Karanth, N G; Umesh-Kumar, S

    2000-04-14

    A 125-kDa starch hydrolysing enzyme of Aspergillus niger characterised by its ability to dextrinise and saccharify starch [Suresh et al. (1999) Appl. Microbiol. Biotechnol. 51, 673-675] was also found to possess activity towards raw starch. Segregation of these activities in the 71-kDa glucoamylase and a 53-kDa alpha-amylase-like enzyme supported by antibody cross-reactivity studies and the isolation of mutants based on assay screens for the secretion of particular enzyme forms revealed the 125-kDa starch hydrolysing enzyme as their precursor. N-terminal sequence analysis further revealed that the 71-kDa glucoamylase was the N-terminal product of the precursor enzyme. Immunological cross reactivity of the 53-kDa amylase with antibodies raised against the precursor enzyme but not with the 71- and 61-kDa glucoamylase antibodies suggested that this enzyme activity is represented by the C-terminal fragment of the precursor. The N-terminal sequence of the 53-kDa protein showed similarity to the reported Taka amylase of Aspergillus oryzae. Antibody cross-reactivity to a 10-kDa non-enzymic peptide and a 61-kDa glucoamylase described these proteins as products of the 71-kDa glucoamylase. Identification of only the precursor starch hydrolysing enzyme in the protein extracts of fungal protoplasts suggested proteolytic processing in the cellular periplasmic space as the cause for the secretion of multiple forms of amylases by A. niger. PMID:10767433

  10. Uterine Endoplasmic Reticulum Stress and Its Unfolded Protein Response May Regulate Caspase 3 Activation in the Pregnant Mouse Uterus

    PubMed Central

    Suresh, Arvind; Subedi, Kalpana; Kyathanahalli, Chandrashekara; Jeyasuria, Pancharatnam; Condon, Jennifer C.

    2013-01-01

    We have previously proposed that uterine caspase-3 may modulate uterine contractility in a gestationally regulated fashion. The objective of this study was to determine the mechanism by which uterine caspase-3 is activated and consequently controlled in the pregnant uterus across gestation. Utilizing the mouse uterus as our gestational model we examined the intrinsic and extrinsic apoptotic signaling pathways and the endoplasmic reticulum stress response as potential activators of uterine caspase-3 at the transcriptional and translational level. Our study revealed robust activation of the uterine myocyte endoplasmic reticulum stress response and its adaptive unfolded protein response during pregnancy coinciding respectively with increased uterine caspase-3 activity and its withdrawal to term. In contrast the intrinsic and extrinsic apoptotic signaling pathways remained inactive across gestation. We speculate that physiological stimuli experienced by the pregnant uterus likely potentiates the uterine myocyte endoplasmic reticulum stress response resulting in elevated caspase-3 activation, which is isolated to the pregnant mouse myometrium. However as term approaches, activation of an elevated adaptive unfolded protein response acts to limit the endoplasmic reticulum stress response inhibiting caspase-3 resulting in its decline towards term. We speculate that these events have the capacity to regulate gestational length in a caspase-3 dependent manner. PMID:24058658

  11. CRP-dependent positive autoregulation and proteolytic degradation regulate competence activator Sxy of Escherichia coli.

    PubMed

    Jaskólska, Milena; Gerdes, Kenn

    2015-03-01

    Natural competence, the ability of bacteria to take up exogenous DNA and incorporate it into their chromosomes, is in most bacteria a transient phenomenon under complex genetic and environmental control. In the Gram-negative bacteria Haemophilus influenzae and Vibrio cholerae, the master regulator Sxy/TfoX controls competence development. Although not known to be naturally competent, Escherichia coli possesses a Sxy homologue and a competence regulon containing the genes required for DNA uptake. Here, we show that in contrast to other characterised Gamma-proteobacteria, E. coli Sxy is positively autoregulated at the level of transcription by a mechanism that requires cAMP receptor protein (CRP), cyclic AMP (cAMP) and a CRP-S site in the sxy promoter. Similarly, we found no evidence that Sxy expression in E. coli was regulated at the translational level. However, our analysis revealed that Sxy is an unstable protein and that its cellular level is negatively regulated at the post-translational level via degradation by Lon protease. Interestingly, in the Gram-positive model organism Bacillus subtilis, the competence master regulator ComK is also positively autoregulated at the level of transcription and negatively regulated by proteolysis. Together, these findings reveal striking similarities between the competence regulons of a Gram-positive and a Gram-negative bacterium. PMID:25491382

  12. Azocasein Substrate for Determination of Proteolytic Activity: Reexamining a Traditional Method Using Bromelain Samples.

    PubMed

    Coêlho, Diego F; Saturnino, Thais Peron; Fernandes, Fernanda Freitas; Mazzola, Priscila Gava; Silveira, Edgar; Tambourgi, Elias Basile

    2016-01-01

    Given the importance of protease's worldwide market, the determination of optimum conditions and the development of a standard protocol are critical during selection of a reliable method to determine its bioactivity. This paper uses quality control theory to validate a modified version of a method proposed by Charney and Tomarelli in 1947. The results obtained showed that using azocasein substrate bromelain had its optimum at 45°C and pH 9 (Glycine-NaOH 100 mM). We also quantified the limit of detection (LoD) and limit of quantification (LoQ) in the above-mentioned optimum (0.072 and 0.494 mg·mL(-1) of azocasein, resp.) and a calibration curve that correlates optical density with the amount of substrate digested. In all analysed samples, we observed a significant decrease in response after storage (around 17%), which suggests its use must be immediately after preparation. Thus, the protocol presented in this paper offers a significant improvement, given that subjective definitions are commonly used in the literature and this simple mathematical approach makes it clear and concise. PMID:26925415

  13. Azocasein Substrate for Determination of Proteolytic Activity: Reexamining a Traditional Method Using Bromelain Samples

    PubMed Central

    Mazzola, Priscila Gava

    2016-01-01

    Given the importance of protease's worldwide market, the determination of optimum conditions and the development of a standard protocol are critical during selection of a reliable method to determine its bioactivity. This paper uses quality control theory to validate a modified version of a method proposed by Charney and Tomarelli in 1947. The results obtained showed that using azocasein substrate bromelain had its optimum at 45°C and pH 9 (Glycine-NaOH 100 mM). We also quantified the limit of detection (LoD) and limit of quantification (LoQ) in the above-mentioned optimum (0.072 and 0.494 mg·mL−1 of azocasein, resp.) and a calibration curve that correlates optical density with the amount of substrate digested. In all analysed samples, we observed a significant decrease in response after storage (around 17%), which suggests its use must be immediately after preparation. Thus, the protocol presented in this paper offers a significant improvement, given that subjective definitions are commonly used in the literature and this simple mathematical approach makes it clear and concise. PMID:26925415

  14. Fermentation of quinoa and wheat slurries by Lactobacillus plantarum CRL 778: proteolytic activity.

    PubMed

    Dallagnol, Andrea Micaela; Pescuma, Micaela; De Valdez, Graciela Font; Rollán, Graciela

    2013-04-01

    Quinoa fermentation by lactic acid bacteria (LAB) is an interesting alternative to produce new bakery products with high nutritional value; furthermore, they are suitable for celiac patients because this pseudo-cereal contains no gluten. Growth and lactic acid production during slurry fermentations by Lactobacillus plantarum CRL 778 were greater in quinoa (9.8 log cfu/mL, 23.1 g/L) than in wheat (8.9 log cfu/mL, 13.9 g/L). Lactic fermentation indirectly stimulated flour protein hydrolysis by endogenous proteases of both slurries. However, quinoa protein hydrolysis was faster, reaching 40-100% at 8 h of incubation, while wheat protein hydrolysis was only 0-20%. In addition, higher amounts of peptides (24) and free amino acids (5 g/L) were determined in quinoa compared to wheat. Consequently, greater concentrations (approx. 2.6-fold) of the antifungal compounds (phenyllactic and hydroxyphenyllactic acids) were synthesized from Phe and Tyr in quinoa by L. plantarum CRL 778, an antifungal strain. These promising results suggest that this LAB strain could be used in the formulation of quinoa sourdough to obtain baked goods with improved nutritional quality and shelf life, suitable for celiac patients. PMID:23129182

  15. In Vitro Proteolytic Inhibition, Polyphenol Oxidase Activity, and Soluble O-Diphenols in Grasses and Cereals

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Harvesting and storing high-quality forage in the cool humid regions of agricultural production remains a challenge due to the potentially high degree of protein degradation during ensiling. Red clover is an exception with high protein levels at harvest that are maintained during ensiling. Decreased...

  16. Effect of dietary organic selenium on muscle proteolytic activity and water-holding capacity in pork.

    PubMed

    Calvo, L; Toldrá, F; Aristoy, M C; López-Bote, C J; Rey, A I

    2016-11-01

    This study evaluates the effect of dietary selenium (Se) supplementation source (organic, Se-enriched yeast; SY vs. inorganic, sodium selenite; SS), dose (0.2: L vs. 0.4: H mg/kg) and the combination of Se and vitamin E (VITE+SS) for 26days on drip loss, TBARS, colour changes, myofibrillar protein pattern and proteolysis in pork. The lowest water losses were observed in the SY-H group when compared to the others. SY-H and VITE+SS groups presented lower myofibrillar protein hydrolysis/oxidation. VITE+SS supplementation also resulted in higher PRO, TRP and PHE content at days 2 and 7, whereas the SY group showed increased GLY and CAR and tended to have higher TAU and ANS at day 2. The myofibrillar fragmentation index was not modified by the dietary treatment; however, at day 8, it tended to be higher in groups supplemented with SeY and VITE+SS. The results of the present study might indicate a possible relation between muscle proteolysis and water loss. PMID:27232379

  17. [Proteolytic enzymes in the prevention and treatment of early ventilatory complications following lung resection in patients with tuberculosis].

    PubMed

    Ershov, V N; Krylov, V V; Zolotarev, V P

    1989-01-01

    The authors examined 259 patients with tuberculosis of the lungs before and after partial resection of the lungs. Proteolytic enzymes were administered endotracheally in the pre- and postoperative periods to 132 patients (main group), 127 patients (control group) received a placebo. Endotracheal administration of proteolytic enzymes in the main group reduced the number of early pleuropulmonary complications of the type of atelectasis and hypoventilation by half to one third and accelerated their resolution by 3-4 times as compared to these values in the control group. Fibrous changes and pleural adhesions on the side of the operation formed from the early pleuropulmonary complications more often in the control than in the main group. The function of external respiration was restored under the effect of the proteolytic enzymes in the control group 1-2 months after segmental and combined resection, in the control group it was restored 3-4 months and later after the operation. PMID:2687135

  18. Effects on fibrinogen, fibrin, and blood coagulation of proteolytic extracts from fruits of Pseudananas macrodontes, Bromelia balansae, and B. hieronymi (Bromeliaceae) in comparison with bromelain.

    PubMed

    Errasti, María E; Prospitti, Anabela; Viana, Carolina A; Gonzalez, Mariana M; Ramos, Márcio V; Rotelli, Alejandra E; Caffini, Néstor O

    2016-06-01

    Extracts rich in cysteine proteases obtained from fruits of Pseudananas macrodontes (Pm), Bromelia balansae (Bb), and B. hieronymi (Bh) have previously shown an anti-inflammatory effect on animal models. Given the close relationship between hemostasis and inflammation, it is attractive to investigate therapeutic agents capable of modulating both systems. The aim of this work was to study the effect of Pm, Bb, and Bh on fibrin(ogen) and blood coagulation compared with stem bromelain (Bro). Action on fibrinogen was electrophoretically and spectrophotometrically evaluated, fibrinolytic activity was measured both electrophoretically and by the fibrin plate assay, and the effect on blood coagulation was studied by conventional coagulation tests (PT and APPT). All extracts showed the same proteolytic preference for fibrinogen subunits, that is Aα > Bβ, whereas γ was partially hydrolyzed by 100-fold concentration increase. Unlike Bro, cysteine proteases of Pm, Bb, and Bh increased absorbance at 540 nm of fibrinogen solution, suggesting thrombin-like activity, which was time-dependent and reached maximum values at lower concentration. All extracts showed the same proteolytic preference for fibrin subunits; however Pm, Bb, and Bh showed lower fibrinolytic activity than Bro at the assayed concentrations. Although Bb acted only as anticoagulant, Pm, Bh, and unexpectedly Bro showed dual action on blood coagulation: at low concentration showed procoagulant effect and at high concentration anticoagulant effect. Results reveal new plant species as potential sources of pharmacological agents for the treatment of a wide range of hemostatic disorders as well as to wound healing. PMID:26886361

  19. Host cell killing by the West Nile Virus NS2B-NS3 proteolytic complex: NS3 alone is sufficient to recruit caspase-8-based apoptotic pathway

    SciTech Connect

    Ramanathan, Mathura P.; Chambers, Jerome A.; Pankhong, Panyupa; Chattergoon, Michael; Attatippaholkun, Watcharee; Dang, Kesen; Shah, Neelima; Weiner, David B. . E-mail: dbweiner@mail.med.upenn.edu

    2006-02-05

    The West Nile Virus (WNV) non-structural proteins 2B and 3 (NS2B-NS3) constitute the proteolytic complex that mediates the cleavage and processing of the viral polyprotein. NS3 recruits NS2B and NS5 proteins to direct protease and replication activities. In an effort to investigate the biology of the viral protease, we cloned cDNA encoding the NS2B-NS3 proteolytic complex from brain tissue of a WNV-infected dead crow, collected from the Lower Merion area (Merion strain). Expression of the NS2B-NS3 gene cassette induced apoptosis within 48 h of transfection. Electron microscopic analysis of NS2B-NS3-transfected cells revealed ultra-structural changes that are typical of apoptotic cells including membrane blebbing, nuclear disintegration and cytoplasmic vacuolations. The role of NS3 or NS2B in contributing to host cell apoptosis was examined. NS3 alone triggers the apoptotic pathways involving caspases-8 and -3. Experimental results from the use of caspase-specific inhibitors and caspase-8 siRNA demonstrated that the activation of caspase-8 was essential to initiate apoptotic signaling in NS3-expressing cells. Downstream of caspase-3 activation, we observed nuclear membrane ruptures and cleavage of the DNA-repair enzyme, PARP in NS3-expressing cells. Nuclear herniations due to NS3 expression were absent in the cells treated with a caspase-3 inhibitor. Expression of protease and helicase domains themselves was sufficient to trigger apoptosis generating insight into the apoptotic pathways triggered by NS3 from WNV.

  20. Proteolytic inactivation of alpha-1-antitrypsin by human neutrophils: involvement of multiple and interlinked cell responses to phagocytosable targets.

    PubMed

    Ottonello, L; Dapino, P; Scirocco, M; Dallegri, F; Sacchetti, C

    1994-01-01

    Neutrophil polymorphonuclear leukocytes (PMN) can inactivate the PMN-elastase inhibitor alpha-1-antitrypsin (A1AT) proteolytically, by using metalloproteinases normally stored as zymogens in myeloperoxidase (MPO)-negative granules. Supernatants from opsonized zymosan (OPZ)-triggered human PMN cleaved and inactivated human A1AT through a process inhibitable by metal-chelators, suggesting that the interaction of PMN with OPZ leads to the extracellular availability of active metalloenzymes. During OPZ-triggering, PMN used approximately 80% of the generated hydrogen peroxide (H2O2) to produce HOCl by means of the MPO pathway, while the remainder was catabolized by PMN themselves. No H2O2 was available as free compound in the extracellular environment and hydroxyl (.OH) or .OH-like radicals were not generated. The selective deletion of single components of the HOCl-generating MPO pathway resulted in the generation of PMN supernatants free of active metalloenzymes but rich of the corresponding zymogens. Similar results were obtained by replacing normal PMN with cells from a patient with hereditary MPO deficiency. No evidence was obtained for the intervention or contribution of .OH-like radicals, serine-proteinases and oxidized glutathione in the transformation of the zymogens into enzymes able to inactivate A1AT. On concluding, PMN undergoing phagocytosis release MPO in amount sufficient to handle the extracellular pool of the generated H2O2 entirely, leading to the generation of equimolar amounts of HOCl. In turn, HOCl or a similar compound derived from it interacts with concomitantly released metallozymogens, switching on their A1AT inactivating potential without the apparent contribution of other PMN-derived molecules.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8187807

  1. Monitoring stepwise proteolytic degradation of peptides by supramolecular domino tandem assays and mass spectrometry for trypsin and leucine aminopeptidase.

    PubMed

    Ghale, Garima; Kuhnert, Nikolai; Nau, Werner M

    2012-03-01

    A label-free optical detection method has been designed that allows direct monitoring of enzymatic peptide digestion in vitro. The method is based on the addition of a reporter pair, composed of the macrocyclic host cucurbit[7]uril (CB7) and the fluorescent dye acridine orange (AO), to detect the proteolytic degradation of peptides. The enzymatic activity of trypsin and leucine aminopeptidase (LAP) was investigated using H-LSRFSWGA-OH as a substrate. The substrate as well as the intermediary and final products (i.e., H-FSWGA-OH and phenylalanine) formed during its enzymatic hydrolysis differ in their binding affinity to the receptor CB7, which results in varying degrees of dye displacement and, therefore, different fluorescence intensities. CB7 showed a relatively weak binding constant of K approximately 10(4) M(-1) with the substrate, a relatively strong binding constant of K > or = 10(6) M(-1) with H-FSWGA-OH (which is a final product formed by trypsin digestion and the intermediary product formed during the enzymatic activity of LAP), and a moderate binding constant of K < or = 10(5) M(-1) with phenylalanine. Owing to this differential binding affinity of CB7 with the substrate and the corresponding products, the digestion of a peptide by trypsin was followed as a decrease in fluorescence signal, while the complete degradation of the peptide by LAP was monitored as a decrease and a subsequent increase in fluorescence signal. The k(cat)/K(M) value for trypsin (2.0 x 10(7) min(-1) M(-1)) was derived from the change in fluorescence signal with time. Additionally, the complete degradation of the peptide by LAP was also followed by mass spectrometry. The use of a supramolecular sensing ensemble (macrocyclic host and dye) as a fluorescent reporter pair gives this method the flexibility to adapt for monitoring the stepwise degradation of different biologically relevant peptides by other proteases. PMID:22545408

  2. What Are the Proteolytic Enzymes of Honey and What They Do Tell Us? A Fingerprint Analysis by 2-D Zymography of Unifloral Honeys

    PubMed Central

    Rossano, Rocco; Larocca, Marilena; Polito, Teresa; Perna, Anna Maria; Padula, Maria Carmela; Martelli, Giuseppe; Riccio, Paolo

    2012-01-01

    Honey is a sweet and healthy food produced by honeybees (Apis mellifera L.) from flower nectars. Using bidimensional zymography, we have detected the, until now unrevealed, proteolytic activities present in row honey samples. The resulting zymograms were specific for each type of the four unifloral honey under study, and enzymes were identified as serine proteases by the use of specific inhibitors. Further, using bidimensional electrophoresis, we have shown that honey proteases are able to degrade the major Royal Jelly proteins and in particular MRPJ-1, the protein that promotes queen differentiation in honeybees. Our findings open new perspectives for the better understanding of honeybee development, social behaviour and role in honey production. The now discovered honey proteases may influence honey properties and quality, and bidimensional zymograms might be useful to distinguish between different honey types, establish their age and floral origin, and allow honey certification. PMID:23145107

  3. Sadaaki Iwanaga: Discovery of the lipopolysaccharide- and beta-1,3-D-glucan-mediated proteolytic cascade and unique proteins in invertebrate immunity.

    PubMed

    Kawabata, Shun-ichiro; Muta, Tatsushi

    2010-05-01

    Horseshoe crab haemolymph contains a single type of cells, granular haemocytes, which are extremely sensitive to bacterial lipopolysaccharides (LPS) and lead to haemolymph coagulation. Sadaaki Iwanaga isolated protease zymogens from the haemocytes and reconstituted LPS and beta-1,3-d-glucans-mediated haemolymph coagulation. This led to the first discovery of a proteolytic cascade triggered by pathogen-associated molecular patterns, an important milestone for studies on invertebrate innate immunity. Moreover, he separated components derived from haemocyte granules and haemolymph plasma, and consequently identified unique defense molecules, such as lectins, serpins, cystatins, antimicrobial substances and substrates for transglutaminase. Through steady and persistent studies on the horseshoe crab host defense system, he made great progress in the field. Now we know that LPS-induced haemocyte exocytosis leads not only to coagulation but also activates a sophisticated immune response network that coordinately induces pathogen recognition, elimination and wound healing. PMID:20406733

  4. The effects of Capn1 gene inactivation on skeletal muscle growth, development, and atrophy, and the compensatory role of other proteolytic systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Myofibrillar protein turnover is a key component of muscle growth and degeneration, requiring proteolytic enzymes to degrade the skeletal muscle proteins. The objective of this study was to investigate the role of the calpain proteolytic system in muscle growth development using µ-calpain knockout (...

  5. Sequencing Lys-N proteolytic peptides by ESI and MALDI tandem mass spectrometry.

    PubMed

    Dupré, Mathieu; Cantel, Sonia; Verdié, Pascal; Martinez, Jean; Enjalbal, Christine

    2011-02-01

    In this study, we explored the MS/MS behavior of various synthetic peptides that possess a lysine residue at the N-terminal position. These peptides were designed to mimic peptides produced upon proteolysis by the Lys-N enzyme, a metalloendopeptidase issued from a Japanese fungus Grifola frondosa that was recently investigated in proteomic studies as an alternative to trypsin digestion, as a specific cleavage at the amide X-Lys chain is obtained that provides N-terminal lysine peptide fragments. In contrast to tryptic peptides exhibiting a lysine or arginine residue solely at the C-terminal position, and are thus devoid of such basic amino acids within the sequence, these Lys-N proteolytic peptides can contain the highly basic arginine residue anywhere within the peptide chain. The fragmentation patterns of such sequences with the ESI-QqTOF and MALDI-TOF/TOF mass spectrometers commonly used in proteomic bottom-up experiments were investigated. PMID:21472586

  6. Sequencing Lys-N Proteolytic Peptides by ESI and MALDI Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Dupré, Mathieu; Cantel, Sonia; Verdié, Pascal; Martinez, Jean; Enjalbal, Christine

    2011-02-01

    In this study, we explored the MS/MS behavior of various synthetic peptides that possess a lysine residue at the N-terminal position. These peptides were designed to mimic peptides produced upon proteolysis by the Lys-N enzyme, a metalloendopeptidase issued from a Japanese fungus Grifola frondosa that was recently investigated in proteomic studies as an alternative to trypsin digestion, as a specific cleavage at the amide X-Lys chain is obtained that provides N-terminal lysine peptide fragments. In contrast to tryptic peptides exhibiting a lysine or arginine residue solely at the C-terminal position, and are thus devoid of such basic amino acids within the sequence, these Lys-N proteolytic peptides can contain the highly basic arginine residue anywhere within the peptide chain. The fragmentation patterns of such sequences with the ESI-QqTOF and MALDI-TOF/TOF mass spectrometers commonly used in proteomic bottom-up experiments were investigated.

  7. Mitochondrial AAA proteases--towards a molecular understanding of membrane-bound proteolytic machines.

    PubMed

    Gerdes, Florian; Tatsuta, Takashi; Langer, Thomas

    2012-01-01

    Mitochondrial AAA proteases play an important role in the maintenance of mitochondrial proteostasis. They regulate and promote biogenesis of mitochondrial proteins by acting as processing enzymes and ensuring the selective turnover of misfolded proteins. Impairment of AAA proteases causes pleiotropic defects in various organisms including neurodegeneration in humans. AAA proteases comprise ring-like hexameric complexes in the mitochondrial inner membrane and are functionally conserved from yeast to man, but variations are evident in the subunit composition of orthologous enzymes. Recent structural and biochemical studies revealed how AAA proteases degrade their substrates in an ATP dependent manner. Intersubunit coordination of the ATP hydrolysis leads to an ordered ATP hydrolysis within the AAA ring, which ensures efficient substrate dislocation from the membrane and translocation to the proteolytic chamber. In this review, we summarize recent findings on the molecular mechanisms underlying the versatile functions of mitochondrial AAA proteases and their relevance to those of the other AAA+ machines. PMID:22001671

  8. Fibrin Clots Are Equilibrium Polymers That Can Be Remodeled Without Proteolytic Digestion

    NASA Astrophysics Data System (ADS)

    Chernysh, Irina N.; Nagaswami, Chandrasekaran; Purohit, Prashant K.; Weisel, John W.

    2012-11-01

    Fibrin polymerization is a necessary part of hemostasis but clots can obstruct blood vessels and cause heart attacks and strokes. The polymerization reactions are specific and controlled, involving strong knob-into-hole interactions to convert soluble fibrinogen into insoluble fibrin. It has long been assumed that clots and thrombi are stable structures until proteolytic digestion. On the contrary, using the technique of fluorescence recovery after photobleaching, we demonstrate here that there is turnover of fibrin in an uncrosslinked clot. A peptide representing the knobs involved in fibrin polymerization can compete for the holes and dissolve a preformed fibrin clot, or increase the fraction of soluble oligomers, with striking rearrangements in clot structure. These results imply that in vivo clots or thrombi are more dynamic structures than previously believed that may be remodeled as a result of local environmental conditions, may account for some embolization, and suggest a target for therapeutic intervention.

  9. Lipoprotein receptors and cholesterol in APP trafficking and proteolytic processing, implications for Alzheimer's disease.

    PubMed

    Marzolo, Maria-Paz; Bu, Guojun

    2009-04-01

    Amyloid-beta (Abeta) peptide accumulation in the brain is central to the pathogenesis of Alzheimer's disease (AD). Abeta is produced through proteolytic processing of a transmembrane protein, beta-amyloid precursor protein (APP), by beta- and gamma-secretases. Mounting evidence has demonstrated that alterations in APP cellular trafficking and localization directly impact its processing to Abeta. Members of the low-density lipoprotein receptor family, including LRP, LRP1B, SorLA/LR11, and apoER2, interact with APP and regulate its endocytic trafficking. Additionally, APP trafficking and processing are greatly affected by cellular cholesterol content. In this review, we summarize the current understanding of the roles of lipoprotein receptors and cholesterol in APP trafficking and processing and their implication for AD pathogenesis and therapy. PMID:19041409

  10. Determinants of Affinity and Proteolytic Stability in Interactions of Kunitz Family Protease Inhibitors with Mesotrypsin

    SciTech Connect

    M Salameh; A Soares; D Navaneetham; D Sinha; P Walsh; E Radisky

    2011-12-31

    An important functional property of protein protease inhibitors is their stability to proteolysis. Mesotrypsin is a human trypsin that has been implicated in the proteolytic inactivation of several protein protease inhibitors. We have found that bovine pancreatic trypsin inhibitor (BPTI), a Kunitz protease inhibitor, inhibits mesotrypsin very weakly and is slowly proteolyzed, whereas, despite close sequence and structural homology, the Kunitz protease inhibitor domain of the amyloid precursor protein (APPI) binds to mesotrypsin 100 times more tightly and is cleaved 300 times more rapidly. To define features responsible for these differences, we have assessed the binding and cleavage by mesotrypsin of APPI and BPTI reciprocally mutated at two nonidentical residues that make direct contact with the enzyme. We find that Arg at P{sub 1} (versus Lys) favors both tighter binding and more rapid cleavage, whereas Met (versus Arg) at P'{sub 2} favors tighter binding but has minimal effect on cleavage. Surprisingly, we find that the APPI scaffold greatly enhances proteolytic cleavage rates, independently of the binding loop. We draw thermodynamic additivity cycles analyzing the interdependence of P{sub 1} and P'{sub 2} substitutions and scaffold differences, finding multiple instances in which the contributions of these features are nonadditive. We also report the crystal structure of the mesotrypsin-APPI complex, in which we find that the binding loop of APPI displays evidence of increased mobility compared with BPTI. Our data suggest that the enhanced vulnerability of APPI to mesotrypsin cleavage may derive from sequence differences in the scaffold that propagate increased flexibility and mobility to the binding loop.

  11. High stability of self-assembled peptide nanowires against thermal, chemical, and proteolytic attacks.

    PubMed

    Ryu, Jungki; Park, Chan Beum

    2010-02-01

    Understanding the self-assembly of peptides into ordered nanostructures is recently getting much attention since it can provide an alternative route for fabricating novel bio-inspired materials. In order to realize the potential of the peptide-based nanofabrication technology, however, more information is needed regarding the integrity or stability of peptide nanostructures under the process conditions encountered in their applications. In this study, we investigated the stability of self-assembled peptide nanowires (PNWs) and nanotubes (PNTs) against thermal, chemical, proteolytic attacks, and their conformational changes upon heat treatment. PNWs and PNTs were grown by the self-assembly of diphenylalanine (Phe-Phe), a peptide building block, on solid substrates at different chemical atmospheres and temperatures. The incubation of diphenylalanine under aniline vapor at 150 degrees C led to the formation of PNWs, while its incubation with water vapor at 25 degrees C produced PNTs. We analyzed the stability of peptide nanostructures using multiple tools, such as electron microscopy, thermal analysis tools, circular dichroism, and Fourier-transform infrared spectroscopy. Our results show that PNWs are highly stable up to 200 degrees C and remain unchanged when incubated in aqueous solutions (from pH 1 to 14) or in various chemical solvents (from polar to non-polar). In contrast, PNTs started to disintegrate even at 100 degrees C and underwent a conformational change at an elevated temperature. When we further studied their resistance to a proteolytic environment, we discovered that PNWs kept their initial structure while PNTs fully disintegrated. We found that the high stability of PNWs originates from their predominant beta-sheet conformation and the conformational change of diphenylalanine nanostructures. Our study suggests that self-assembled PNWs are suitable for future nano-scale applications requiring harsh processing conditions. PMID:19777585

  12. Determinants of Affinity and Proteolytic Stability in Interactions of Kunitz Family Protease Inhibitors with Mesotrypsin

    SciTech Connect

    Salameh, M.A.; Soares, A.; Navaneetham, D.; Sinha, D.; Walsh, P. N.; Radisky, E. S.

    2010-11-19

    An important functional property of protein protease inhibitors is their stability to proteolysis. Mesotrypsin is a human trypsin that has been implicated in the proteolytic inactivation of several protein protease inhibitors. We have found that bovine pancreatic trypsin inhibitor (BPTI), a Kunitz protease inhibitor, inhibits mesotrypsin very weakly and is slowly proteolyzed, whereas, despite close sequence and structural homology, the Kunitz protease inhibitor domain of the amyloid precursor protein (APPI) binds to mesotrypsin 100 times more tightly and is cleaved 300 times more rapidly. To define features responsible for these differences, we have assessed the binding and cleavage by mesotrypsin of APPI and BPTI reciprocally mutated at two nonidentical residues that make direct contact with the enzyme. We find that Arg at P{sub 1} (versus Lys) favors both tighter binding and more rapid cleavage, whereas Met (versus Arg) at P'{sub 2} favors tighter binding but has minimal effect on cleavage. Surprisingly, we find that the APPI scaffold greatly enhances proteolytic cleavage rates, independently of the binding loop. We draw thermodynamic additivity cycles analyzing the interdependence of P1 and P'{sub 2} substitutions and scaffold differences, finding multiple instances in which the contributions of these features are nonadditive. We also report the crystal structure of the mesotrypsin {center_dot} APPI complex, in which we find that the binding loop of APPI displays evidence of increased mobility compared with BPTI. Our data suggest that the enhanced vulnerability of APPI to mesotrypsin cleavage may derive from sequence differences in the scaffold that propagate increased flexibility and mobility to the binding loop.

  13. Proteolytic cleavage of Ser52Pro variant transthyretin triggers its amyloid fibrillogenesis

    PubMed Central

    Mangione, P. Patrizia; Porcari, Riccardo; Gillmore, Julian D.; Pucci, Piero; Monti, Maria; Porcari, Mattia; Giorgetti, Sofia; Marchese, Loredana; Raimondi, Sara; Serpell, Louise C.; Chen, Wenjie; Relini, Annalisa; Marcoux, Julien; Clatworthy, Innes R.; Taylor, Graham W.; Tennent, Glenys A.; Robinson, Carol V.; Hawkins, Philip N.; Stoppini, Monica; Wood, Stephen P.; Pepys, Mark B.; Bellotti, Vittorio

    2014-01-01

    The Ser52Pro variant of transthyretin (TTR) produces aggressive, highly penetrant, autosomal-dominant systemic amyloidosis in persons heterozygous for the causative mutation. Together with a minor quantity of full-length wild-type and variant TTR, the main component of the ex vivo fibrils was the residue 49-127 fragment of the TTR variant, the portion of the TTR sequence that previously has been reported to be the principal constituent of type A, cardiac amyloid fibrils formed from wild-type TTR and other TTR variants [Bergstrom J, et al. (2005) J Pathol 206(2):224–232]. This specific truncation of Ser52Pro TTR was generated readily in vitro by limited proteolysis. In physiological conditions and under agitation the residue 49-127 proteolytic fragment rapidly and completely self-aggregates into typical amyloid fibrils. The remarkable susceptibility to such cleavage is likely caused by localized destabilization of the β-turn linking strands C and D caused by loss of the wild-type hydrogen-bonding network between the side chains of residues Ser52, Glu54, Ser50, and a water molecule, as revealed by the high-resolution crystallographic structure of Ser52Pro TTR. We thus provide a structural basis for the recently hypothesized, crucial pathogenic role of proteolytic cleavage in TTR amyloid fibrillogenesis. Binding of the natural ligands thyroxine or retinol-binding protein (RBP) by Ser52Pro variant TTR stabilizes the native tetrameric assembly, but neither protected the variant from proteolysis. However, binding of RBP, but not thyroxine, inhibited subsequent fibrillogenesis. PMID:24474780

  14. Investigation of the types and characteristics of the proteolytic enzymes formed by diverse strains of Proteus species.

    PubMed

    Senior, B W

    1999-07-01

    Many diverse clinical isolates of Proteus mirabilis (48 strains), P. penneri (25), P. vulgaris biogroup 2 (48) and P. vulgaris biogroup 3 (21) from man were examined for their ability to produce proteolytic enzymes and the nature and characteristics of the proteases were studied. All the P. penneri isolates, most (94-90%) of the P. mirabilis and P. vulgaris biogroup 2 isolates, but only 71% of the P. vulgaris biogroup 3 isolates, secreted proteolytic enzymes. These were detected most readily at pH 8 with gelatin as substrate. A strong correlation was found between the ability of a strain to form swarming growth and its ability to secrete proteases. Non-swarming isolates invariably appeared to be non-proteolytic. However, some isolates, particularly of P. vulgaris biogroup 3, were non-proteolytic even when they formed swarming growth. Analysis of the secreted enzymes of the different Proteus spp. on polyacrylamide-gelatin gels under various constraints of pH and other factors showed that they were all EDTA-sensitive metalloproteinases. Analysis of the kinetics of production of the proteases revealed the formation of an additional protease of undefined type and function that was cell-associated and formed before the others were secreted. The secreted protease was subsequently modified to two isoforms whose mass (53-46 kDa) varied with the Proteus spp. and the strain. There was no evidence that the secreted proteases of strains of Proteus spp. were of types other than metalloproteinases. PMID:10403412

  15. The caspase proteolytic system in callipyge and normal lambs in longissimus, semimembranosus, and infraspinatus muscles during postmortem storage.

    PubMed

    Kemp, C M; King, D A; Shackelford, S D; Wheeler, T L; Koohmaraie, M

    2009-09-01

    The objective of this experiment was to determine whether the caspase proteolytic system has a role in postmortem tenderization. Six ewes and 6 wethers that were noncarriers and 6 ewes and 6 wethers that were expressing the callipyge gene were used for this study. Caspase activities were determined in LM at 7 different time points during the postmortem storage period: 0 h, 4 h, 8 h, 24 h, 2 d, 7 d, and 21 d and in semimembranosus (SM) and infraspinatus (IS) muscles at 0 h, 8 h, 24 h, and 7 d from callipyge and noncallipyge (normal) lambs. Calpastatin activity was determined at 0 h, 2 d, 7 d, and 21 d and slice shear force measured at 2, 7, and 21 d in the LM. Calpastatin activity and slice shear force were greater in LM from callipyge lambs than normal lambs at each time point (P < 0.001 and P < 0.0001, respectively). Caspases 3 and 7 are executioner caspases, and their combined activity was found to decrease during the postmortem storage period in LM, SM, and IS muscles from callipyge and normal lambs. Similarly, activity of the initiator caspase (caspase 9) decreased (P < 0.05) in all 3 muscles across the postmortem storage period in callipyge and normal lambs, and its decrease in activity preceded that of the executioner caspases 3/7. A positive relationship also was detected between caspase 9 and caspase 3/7 in LM, SM, and IS muscles (P < 0.0001, r = 0.85, r = 0.86, r = 0.84, respectively), which is consistent with caspase 9 being responsible for the cleavage and activation of the executioner caspases (caspase 3/7) downstream. Caspase 3/7 and caspase 9 activities at 8 h in SM were greater in normal lamb than callipyge lamb (P < 0.05), with a trend for caspase 3/7 activity to be greater at 24 h postmortem (P = 0.0841). There also was a trend for caspase 3/7 activity to be greater in LM at 21 d in normal lamb than in callipyge lamb (P = 0.053), although there were no differences detected in caspase activities between genotypes in the IS muscle, which is not

  16. The proteolytic fragments of the Alzheimer's disease-associated presenilin-1 form heterodimers and occur as a 100-150-kDa molecular mass complex.

    PubMed

    Capell, A; Grünberg, J; Pesold, B; Diehlmann, A; Citron, M; Nixon, R; Beyreuther, K; Selkoe, D J; Haass, C

    1998-02-01

    Mutations in the presenilin (PS) genes are linked to early onset familial Alzheimer's disease (FAD). PS-1 proteins are proteolytically processed by an unknown protease to two stable fragments of approximately 30 kDa (N-terminal fragment (NTF)) and approximately 20 kDa (C-terminal fragment (CTF)) (Thinakaran, G., Borchelt, D. R., Lee, M. K., Slunt, H. H., Spitzer, L., Kim, G., Ratovitsky, T., Davenport, F., Nordstedt, C., Seeger, M., Hardy, J., Levey, A. I., Gandy, S. E., Jenkins, N. A., Copeland, N. G., Price, D. L., and Sisodia, S. S. (1996) Neuron 17, 181-190). Here we show that the CTF and NTF of PS-1 bind to each other. Fractionating proteins from 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid-extracted membrane preparations by velocity sedimentation reveal a high molecular mass SDS and Triton X-100-sensitive complex of approximately 100-150 kDa. To prove if both proteolytic fragments of PS-1 are bound to the same complex, we performed co-immunoprecipitations using multiple antibodies specific to the CTF and NTF of PS-1. These experiments revealed that both fragments of PS-1 occur as a tightly bound non-covalent complex. Upon overexpression, unclipped wild type PS-1 sediments at a lower molecular weight in glycerol velocity gradients than the endogenous fragments. In contrast, the non-cleavable, FAD-associated PS-1 Deltaexon 9 sediments at a molecular weight similar to that observed for the endogenous proteolytic fragments. This result may indicate that the Deltaexon 9 mutation generates a mutant protein that exhibits biophysical properties similar to the naturally occurring PS-1 fragments. This could explain the surprising finding that the Deltaexon 9 mutation is functionally active, although it cannot be proteolytically processed (Baumeister, R., Leimer, U., Zweckbronner, I., Jakubek, C., Grünberg, J., and Haass, C. (1997) Genes & Function 1, 149-159; Levitan, D., Doyle, T., Brousseau, D., Lee, M., Thinakaran, G., Slunt, H., Sisodia, S., and

  17. A Drosophila model of GSS syndrome suggests defects in active zones are responsible for pathogenesis of GSS syndrome

    PubMed Central

    Choi, Jin-Kyu; Jeon, Yong-Chul; Lee, Dae-Weon; Oh, Jae-Min; Lee, Hyun-Pil; Jeong, Byung-Hoon; Carp, Richard I.; Koh, Young Ho; Kim, Yong-Sun

    2010-01-01

    We have established a Drosophila model of Gerstmann–Sträussler–Scheinker (GSS) syndrome by expressing mouse prion protein (PrP) having leucine substitution at residue 101 (MoPrPP101L). Flies expressing MoPrPP101L, but not wild-type MoPrP (MoPrP3F4), showed severe defects in climbing ability and early death. Expressed MoPrPP101L in Drosophila was differentially glycosylated, localized at the synaptic terminals and mainly present as deposits in adult brains. We found that behavioral defects and early death of MoPrPP101L flies were not due to Caspase 3-dependent programmed cell death signaling. In addition, we found that Type 1 glutamatergic synaptic boutons in larval neuromuscular junctions of MoPrPP101L flies showed significantly increased numbers of satellite synaptic boutons. Furthermore, the amount of Bruchpilot and Discs large in MoPrPP101L flies was significantly reduced. Brains from scrapie-infected mice showed significantly decreased ELKS, an active zone matrix marker compared with those of age-matched control mice. Thus, altered active zone structures at the molecular level may be involved in the pathogenesis of GSS syndrome in Drosophila and scrapie-infected mice. PMID:20829230

  18. Analysis of caspase3 activation in ChanSu-induced apoptosis of ASTC-a-1 cells by fluorescence techniques

    NASA Astrophysics Data System (ADS)

    Sun, Lei; Chen, Tongsheng; Wang, Longxiang; Wang, Huiying

    2008-02-01

    ChanSu(CS), a traditional Chinese medicine, is composed of many chemical compoments. It is isolated from the dried white secretion of the auricular and skin glands of toads, and it has been widely used for treating the heart diseases and other systemic illnesses. However, it is difficult to judge antitumor effect of agents derived from ChanSu and the underlying mechanism of ChanSu inducing cell apoptosis is still unclear. This report was performed to explore the inhibitory effect and mechanism of ChanSu on human lung adenocarcinoma cells (ASTC-a-1). Fluorescence emission spectra and fluorescence resonance energy transfer (FRET) were used to study the caspase-3 activation during the ChanSu-induced human lung adenocarcinoma (ASTC-a-1) cell apoptosis. CCK-8 was used to assay the inhibition of ChanSu on the cell viability. The cells expressing stably with SCAT3 was used to examine if caspase-3 was activated by ChanSu using acceptor photobleaching technique. Our data showed that treatment of ASTC-a-1 cell with ChanSu resulted in the inhibition of viability and induction of apoptosis in a dose-dependent manner and the SCAT3 was almost cleaved 24 h after ChanSu treatment, implying that ChanSu induced cell apoptosis via a caspase-3-dependent death pathway. Our findings extend the knowledge about the cellular signaling mechanisms mediating ChanSu-induced apoptosis.

  19. Identification of Phenolic Compounds from Seed Coats of Differently Colored European Varieties of Pea (Pisum sativum L.) and Characterization of Their Antioxidant and In Vitro Anticancer Activities.

    PubMed

    Stanisavljević, Nemanja S; Ilić, Marija D; Matić, Ivana Z; Jovanović, Živko S; Čupić, Tihomir; Dabić, Dragana Č; Natić, Maja M; Tešić, Živoslav Lj

    2016-01-01

    To date little has been done on identification of major phenolic compounds responsible for anticancer and antioxidant properties of pea (Pisum sativum L.) seed coat extracts. In the present study, phenolic profile of the seed coat extracts from 10 differently colored European varieties has been determined using ultrahigh-performance liquid chromatography-linear trap quadrupole orbitrap mass spectrometer technique. Extracts of dark colored varieties with high total phenolic content (up to 46.56 mg GAE/g) exhibited strong antioxidant activities (measured by 2,2-diphenyl-1-picrylhydrazyl or DPPH assay, and ferric ion reducing and ferrous ion chelating capacity assays) which could be attributed to presence of gallic acid, epigallocatechin, naringenin, and apigenin. The aqueous extracts of dark colored varieties exert concentration-dependent cytotoxic effects on all tested malignant cell lines (human colon adenocarcinoma LS174, human breast carcinoma MDA-MB-453, human lung carcinoma A594, and myelogenous leukemia K562). Correlation analysis revealed that intensities of cytotoxic activity of the extracts strongly correlated with contents of epigallocatechin and luteolin. Cell cycle analysis on LS174 cells in the presence of caspase-3 inhibitor points out that extracts may activate other cell death modalities besides caspase-3-dependent apoptosis. The study provides evidence that seed coat extracts of dark colored pea varieties might be used as potential cancer-chemopreventive and complementary agents in cancer therapy. PMID:27348025

  20. Reduced secretion and altered proteolytic processing caused by missense mutations in progranulin.

    PubMed

    Kleinberger, Gernot; Capell, Anja; Brouwers, Nathalie; Fellerer, Katrin; Sleegers, Kristel; Cruts, Marc; Van Broeckhoven, Christine; Haass, Christian

    2016-03-01

    Progranulin (GRN) is a secreted growth factor involved in various cellular functions, and loss-of-function mutations are a major cause of frontotemporal lobar degeneration (FTLD) with TDP-43 positive pathology. Most FTLD-related GRN mutations are nonsense mutations resulting in reduced GRN expression. Nonsynonymous GRN missense mutations have been described as risk factor for neurodegenerative brain diseases, but their pathogenic nature remains largely elusive. We identified a double missense mutation in GRN leading to amino acid changes p.D33E and p.G35R in an FTLD patient from Turkish origin. Biochemical and cell biological analysis of the double-mutation together with 2 so-far uncharacterized GRN missense mutations (p.C105R and p.V514M) revealed a reduced secretion efficiency of the GRN p.D33E/p.G35R and p.C105R proteins. Furthermore, loss of the conserved cysteine residue affects protein folding and altered proteolytic processing by neutrophil elastase and proteinase 3. Our data indicate that the described variants may cause a loss-of-function, albeit to a lesser extent than GRN null mutations, and hence could be considered as low-penetrant risk factors for neurodegenerative diseases. PMID:26811050

  1. The Impact of Proteolytic Pork Hydrolysate on Microbial, Flavor and Free Amino Acids Compounds of Yogurt.

    PubMed

    Lin, Jinzhong; Hua, Baozhen; Xu, Zhiping; Li, Sha; Ma, Chengjie

    2016-01-01

    The aim of this study was to investigate the influence of proteolytic pork hydrolysate (PPH) on yoghurt production by Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. Fresh lean pork was cut into pieces and mixed with deionized water and dealt with protease, then the resulting PPH was added to milk to investigate the effects of PPH on yoghurt production. The fermentation time, the viable cell counts, the flavor, free amino acids compounds, and sensory evaluation of yoghurt were evaluated. These results showed that PPH significantly stimulated the growth and acidification of the both bacterial strains. When the content of PPH reached 5% (w/w), the increased acidifying rate occurred, which the fermentation time was one hour less than that of the control, a time saving of up to 20% compared with the control. The viable cell counts, the total free amino acids, and the scores of taste, flavor and overall acceptability in PPH-supplemented yoghurt were higher than the control. Furthermore, the contents of some characteristic flavor compounds including acids, alcohols, aldehydes, ketones and esters were richer than the control. We concluded that the constituents of PPH such as small peptide, vitamins, and minerals together to play the stimulatory roles and result in beneficial effect for the yoghurt starter cultures growth. PMID:27621698

  2. Characterization of Protein N-Glycosylation by Analysis of ZIC-HILIC-Enriched Intact Proteolytic Glycopeptides.

    PubMed

    Pohlentz, Gottfried; Marx, Kristina; Mormann, Michael

    2016-01-01

    Zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) solid-phase extraction (SPE) combined with direct-infusion nanoESI mass spectrometry (MS) and tandem MS/MS is a well-suited method for the analysis of protein N-glycosylation. A site-specific characterization of N-glycopeptides is achieved by the combination of proteolytic digestions employing unspecific proteases, glycopeptide enrichment by use of ZIC-HILIC SPE, and subsequent mass spectrometric analysis. The use of thermolysin or a mixture of trypsin and chymotrypsin leads per se to a mass-based separation, that is, small nonglycosylated peptides and almost exclusively glycopeptides at higher m/z values. As a result of their higher hydrophilicity N-glycopeptides comprising short peptide backbones are preferably accumulated by the ZIC-HILIC-based separation procedure. By employing this approach complications associated with low ionization efficiencies of N-glycopeptides resulting from signal suppression in the presence of highly abundant nonglycosylated peptides can be largely reduced. Here, we describe a simple protocol aimed at the enrichment of N-glycopeptides derived from in-solution and in-gel digestions of SDS-PAGE-separated glycoproteins preceding mass spectrometric analysis. PMID:26700048

  3. The Impact of Proteolytic Pork Hydrolysate on Microbial, Flavor and Free Amino Acids Compounds of Yogurt

    PubMed Central

    Lin, Jinzhong; Hua, Baozhen; Xu, Zhiping; Li, Sha; Ma, Chengjie

    2016-01-01

    The aim of this study was to investigate the influence of proteolytic pork hydrolysate (PPH) on yoghurt production by Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. Fresh lean pork was cut into pieces and mixed with deionized water and dealt with protease, then the resulting PPH was added to milk to investigate the effects of PPH on yoghurt production. The fermentation time, the viable cell counts, the flavor, free amino acids compounds, and sensory evaluation of yoghurt were evaluated. These results showed that PPH significantly stimulated the growth and acidification of the both bacterial strains. When the content of PPH reached 5% (w/w), the increased acidifying rate occurred, which the fermentation time was one hour less than that of the control, a time saving of up to 20% compared with the control. The viable cell counts, the total free amino acids, and the scores of taste, flavor and overall acceptability in PPH-supplemented yoghurt were higher than the control. Furthermore, the contents of some characteristic flavor compounds including acids, alcohols, aldehydes, ketones and esters were richer than the control. We concluded that the constituents of PPH such as small peptide, vitamins, and minerals together to play the stimulatory roles and result in beneficial effect for the yoghurt starter cultures growth. PMID:27621698

  4. Proteolytic Isoforms of SPARC Induce Adipose Stromal Cell Mobilization in Obesity.

    PubMed

    Tseng, Chieh; Kolonin, Mikhail G

    2016-01-01

    Adipose stromal cells (ASC) are mesenchymal adipocyte progenitors that reside in the peri-endothelium of fat tissue. ASC mobilization and migration accompany white adipose tissue (WAT) remodeling and pathological conditions. Mechanisms regulating ASC trafficking are largely unknown. We previously reported that binding of the matricellular protein secreted protein acidic and rich in cysteine (SPARC) to β1 integrin on ASC surface induces their motility. Here, we show that SPARC is required for ASC mobilization. We report two SPARC proteolytic isoforms, C-SPARC (lacking the N terminus) and N-SPARC (lacking the C terminus), generated in mesenteric WAT of obese mice. C-SPARC, but not N-SPARC, binds to β1 integrin on ASC, while N-SPARC preferentially binds to the extracellular matrix (ECM) and blocks ECM/integrin interaction. Interestingly, both C-SPARC and N-SPARC induce ASC deadhesion from the ECM, which is associated with modulation of integrin-dependent FAK-ERK signaling and integrin-independent ILK-Akt signaling. We show that these SPARC isoforms, acting on ASC through distinct mechanisms, have an additive effect in inducing ASC migration. PMID:26381424

  5. Complete amino acid sequence of a histidine-rich proteolytic fragment of human ceruloplasmin.

    PubMed

    Kingston, I B; Kingston, B L; Putnam, F W

    1979-04-01

    The complete amino acid sequence has been determined for a fragment of human ceruloplasmin [ferroxidase; iron(II):oxygen oxidoreductase, EC 1.16.3.1]. The fragment (designated Cp F5) contains 159 amino acid residues and has a molecular weight of 18,650; it lacks carbohydrate, is rich in histidine, and contains one free cysteine that may be part of a copper-binding site. This fragment is present in most commercial preparations of ceruloplasmin, probably owing to proteolytic degradation, but can also be obtained by limited cleavage of single-chain ceruloplasmin with plasmin. Cp F5 probably is an intact domain attached to the COOH-terminal end of single-chain ceruloplasmin via a labile interdomain peptide bond. A model of the secondary structure predicted by empirical methods suggests that almost one-third of the amino acid residues are distributed in alpha helices, about a third in beta-sheet structure, and the remainder in beta turns and unidentified structures. Computer analysis of the amino acid sequence has not demonstrated a statistically significant relationship between this ceruloplasmin fragment and any other protein, but there is some evidence for an internal duplication. PMID:287005

  6. Proteolytic processing of Escherichia coli twin-arginine signal peptides by LepB.

    PubMed

    Lüke, Iris; Handford, Jennifer I; Palmer, Tracy; Sargent, Frank

    2009-12-01

    The twin-arginine translocation (Tat) apparatus is a protein targeting system found in the cytoplasmic membranes of many prokaryotes. Substrate proteins of the Tat pathway are synthesised with signal peptides bearing SRRxFLK 'twin-arginine' amino acid motifs. All Tat signal peptides have a common tripartite structure comprising a polar N-terminal region, followed by a hydrophobic region of variable length and a polar C-terminal region. In Escherichia coli, Tat signal peptides are proteolytically cleaved after translocation. The signal peptide C-terminal regions contain conserved AxA motifs, which are possible recognition sequences for leader peptidase I (LepB). In this work, the role of LepB in Tat signal peptide processing was addressed directly. Deliberate repression of lepB expression prevented processing of all Tat substrates tested, including SufI, AmiC, and a TorA-23K reporter protein. In addition, electron microscopy revealed gross defects in cell architecture and membrane integrity following depletion of cellular LepB protein levels. PMID:19809807

  7. Single-molecule protein unfolding and translocation by an ATP-fueled proteolytic machine

    PubMed Central

    Aubin-Tam, Marie-Eve; Olivares, Adrian O.; Sauer, Robert T.; Baker, Tania A.; Lang, Matthew J.

    2011-01-01

    All cells employ ATP-powered proteases for protein-quality control and regulation. In the ClpXP protease, ClpX is a AAA+ machine that recognizes specific protein substrates, unfolds these molecules, and then translocates the denatured polypeptide through a central pore and into ClpP for degradation. Here, we use optical-trapping nanometry to probe the mechanics of enzymatic unfolding and translocation of single molecules of a multidomain substrate. Our experiments demonstrate the capacity of ClpXP and ClpX to perform mechanical work under load, reveal very fast and highly cooperative unfolding of individual substrate domains, suggest a translocation step size of 5–8 amino acids, and support a power-stroke model of denaturation in which successful enzyme-mediated unfolding of stable domains requires coincidence between mechanical pulling by the enzyme and a transient stochastic reduction in protein stability. We anticipate that single-molecule studies of the mechanical properties of other AAA+ proteolytic machines will reveal many shared features with ClpXP. PMID:21496645

  8. Small-molecule-mediated rescue of protein function by an inducible proteolytic shunt

    PubMed Central

    Pratt, Matthew R.; Schwartz, Edmund C.; Muir, Tom W.

    2007-01-01

    Controlling protein function through posttranslational manipulations has emerged as an attractive complementary technology to existing genetic systems. Often these methods involve developing pharmacological agents to probe protein function without the need to generate a unique compound for each protein family. One common strategy uses small molecules that act as chemical inducers of dimerization by mediating the interaction of two proteins. Herein we report the use of a chemical inducer of dimerization for the development of a posttranslational technology for the manipulation of protein function. This system, split ubiquitin for the rescue of function (SURF), places the complementation of genetically split ubiquitin under the control of rapamycin-induced dimerization of FK506-binding protein and FKBP12-rapamycin-binding protein. Before complementation a “degron” dooms a protein of interest for destruction by the proteasome. Addition of rapamycin results in a proteolytic shunt away from degradation by inducing ubiquitin complementation and cleavage of the protein of interest from the degron. Importantly, the native protein is rescued. We characterized this system with firefly luciferase and went on to apply it to members of three important classes of proteins: proteases (caspase-3), kinases (v-Src), and transcription factors (Smad3). This general strategy should allow for inducible rescue of a variety of proteins in such a way that their native structure and function are maintained. PMID:17563385

  9. Proteolytic cleavage of ataxin-7 promotes SCA7 retinal degeneration and neurological dysfunction.

    PubMed

    Guyenet, Stephan J; Mookerjee, Shona S; Lin, Amy; Custer, Sara K; Chen, Sylvia F; Sopher, Bryce L; La Spada, Albert R; Ellerby, Lisa M

    2015-07-15

    The neurodegenerative disorder spinocerebellar ataxia type 7 (SCA7) is caused by a polyglutamine (polyQ) expansion in the ataxin-7 protein, categorizing SCA7 as one member of a large class of heritable neurodegenerative proteinopathies. Cleavage of ataxin-7 by the protease caspase-7 has been demonstrated in vitro, and the accumulation of proteolytic cleavage products in SCA7 patients and mouse models has been identified as an early pathological change. However, it remains unknown whether a causal relationship exists between ataxin-7 proteolysis and in vivo SCA7 disease progression. To determine whether caspase cleavage is a critical event in SCA7 disease pathogenesis, we generated transgenic mice expressing polyQ-expanded ataxin-7 with a second-site mutation (D266N) to prevent caspase-7 proteolysis. When we compared SCA7-D266N mice with SCA7 mice lacking the D266N mutation, we found that SCA7-D266N mice exhibited improved motor performance, reduced neurodegeneration and substantial lifespan extension. Our findings indicate that proteolysis at the D266 caspase-7 cleavage site is an important mediator of ataxin-7 neurotoxicity, suggesting that inhibition of caspase-7 cleavage of polyQ-ataxin-7 may be a promising therapeutic strategy for this untreatable disorder. PMID:25859008

  10. A new insight into phagocytosis of apoptotic cells: proteolytic enzymes divert the recognition and clearance of polymorphonuclear leukocytes by macrophages.

    PubMed

    Guzik, K; Bzowska, M; Smagur, J; Krupa, O; Sieprawska, M; Travis, J; Potempa, J

    2007-01-01

    The recognition of phosphatidylserine (PS) on the surface of any apoptotic cell is considered to be a key event for its clearance. We challenge this concept by showing that pretreatment of neutrophils with either host or bacterial protease affects their uptake by human monocyte-derived macrophages without having an effect on cell-surface PS presentation. Specifically, whereas preincubation of apoptotic neutrophils with cathepsin G or thrombin significantly inhibited their uptake, gingipains R or clostripain enhanced phagocytosis by macrophages. Moreover, bacterial proteinases sensitized healthy neutrophils for uptake by macrophages, whereas endogenous proteinases were unable to elicit this effect. This stimulation was apparently owing to the combined effect of proteolytic cleavage of an antiphagocytic signal (CD31) and the generation of a novel 'eat-me' signal on the neutrophil surface. These results argue that neutrophil recognition and phagocytosis by macrophages is mediated by a protein ligand whose proteolytic modification could affect the local inflammatory process. PMID:16628232

  11. [The permeability of the hemato-encephalic barrier and the proteolytic potential of the cerebrospinal fluid in severe craniocerebral trauma].

    PubMed

    Churliaev, Iu A; Nikiforova, N V; Lutsik, A A; Kuksinskiĭ, V A; Lykova, O F; Martynenkov, V Ia; Karpenko, V S

    1999-01-01

    To study blood-brain barrier permeability and proteolytic changes in in patients with severe brain injury and to evaluate their impact on its course and outcome, the concentrations of albumin, plasminogen (plasmin), alpha 2-macroglobulin, alpha 2-antiplasmin, and alpha 1-antitrypsin were examined in 58 victims by enzyme immunoassay. The control group comprised 20 patients examined for lumbar discal hernia. The studies indicate that early severe brain injury showed blood-brain barrier dysfunction whose severity can be detected by the spinal fluid levels of albumin, plasminogen, and alpha 2-macroglobulin. Proteolytic changes in spinal fluid are determined by its albumin, plasminogen (plasmin), alpha 2-macroglobulin, alpha 2-antiplasmin, and alpha 1-antitrypsin concentrations and affect the development of secondary brain lesion and they are of practical value. PMID:10696680

  12. Effects of reducing fat content on the proteolytic and rheological properties of Cheddar-like caprine milk cheese

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High-moisture Cheddar-like cheeses made from caprine milk containing 3.6, 2.0, 1.0, and 0.1-0.5% fat were manufactured and their proteolytic and rheological properties compared after 1, 3, and 6 mo of aging at 4 deg C. The full-fat (FF), reduced fat (RF), low-fat (LF), and non-fat (NF) cheeses conta...

  13. Effect of culturing conditions on the expression of key enzymes in the proteolytic system of Lactobacillus bulgaricus.

    PubMed

    Hou, Jun-cai; Liu, Fei; Ren, Da-xi; Han, Wei-wei; Du, Yue-ou

    2015-04-01

    The proteolytic system of Lactobacillus bulgaricus breaks down milk proteins into peptides and amino acids, which are essential for the growth of the bacteria. The aim of this study was to determine the expressions of seven key genes in the proteolytic system under different culturing conditions (different phases, initial pH values, temperatures, and nitrogen sources) using real-time polymerase chain reaction (RT-PCR). The transcriptions of the seven genes were reduced by 30-fold on average in the stationary phase compared with the exponential growth phase. The transcriptions of the seven genes were reduced by 62.5-, 15.0-, and 59.0-fold in the strains KLDS 08006, KLDS 08007, and KLDS 08012, respectively, indicating that the expressions of the seven genes were significantly different among strains. In addition, the expressions of the seven genes were repressed in the MRS medium containing casein peptone. The effect of peptone supply on PepX transcription was the weakest compared with the other six genes, and the impact on OppD transcription was the strongest. Moreover, the expressions of the seven genes were significantly different among different strains (P<0.05). All these results indicated that the culturing conditions affected the expression of the proteolytic system genes in Lactobacillus bulgaricus at the transcription level. PMID:25845365

  14. Effect of culturing conditions on the expression of key enzymes in the proteolytic system of Lactobacillus bulgaricus *

    PubMed Central

    Hou, Jun-cai; Liu, Fei; Ren, Da-xi; Han, Wei-wei; Du, Yue-ou

    2015-01-01

    The proteolytic system of Lactobacillus bulgaricus breaks down milk proteins into peptides and amino acids, which are essential for the growth of the bacteria. The aim of this study was to determine the expressions of seven key genes in the proteolytic system under different culturing conditions (different phases, initial pH values, temperatures, and nitrogen sources) using real-time polymerase chain reaction (RT-PCR). The transcriptions of the seven genes were reduced by 30-fold on average in the stationary phase compared with the exponential growth phase. The transcriptions of the seven genes were reduced by 62.5-, 15.0-, and 59.0-fold in the strains KLDS 08006, KLDS 08007, and KLDS 08012, respectively, indicating that the expressions of the seven genes were significantly different among strains. In addition, the expressions of the seven genes were repressed in the MRS medium containing casein peptone. The effect of peptone supply on PepX transcription was the weakest compared with the other six genes, and the impact on OppD transcription was the strongest. Moreover, the expressions of the seven genes were significantly different among different strains (P<0.05). All these results indicated that the culturing conditions affected the expression of the proteolytic system genes in Lactobacillus bulgaricus at the transcription level. PMID:25845365

  15. First description of French V. tubiashii strains pathogenic to mollusk: II. Characterization of properties of the proteolytic fraction of extracellular products.

    PubMed

    Mersni-Achour, Rachida; Imbert-Auvray, Nathalie; Huet, Valérie; Ben Cheikh, Yosra; Faury, Nicole; Doghri, Ibtissem; Rouatbi, Sonia; Bordenave, Stéphanie; Travers, Marie-Agnès; Saulnier, Denis; Fruitier-Arnaudin, Ingrid

    2014-11-01

    Extracellular products (ECPs) of the French Vibrio tubiashii strain 07/118 T2 were previously reported to be toxic for the Pacific oyster Crassostrea gigas. In this study we now assessed host cellular immune responses and bacterial potential effectors by which these ECPs can be associated with host damages. The adhesion capacity (28% inhibition) and phagocytosis ability (56% inhibition) of oyster hemocytes were the main functions affected following in vitro contact between hemocytes and V. tubiashii ECPs. This may be linked to the demonstration of the capability of ECPs to cleave various cellular substrates as oyster collagen. Moreover, a strong metalloproteolytic activity was recorded with general (azocasein) and specific (ADAM) substrates and characterized by the use of standard inhibitors and metal ions. The addition of 1,10-phenanthroline and Zn2+ decreased proteolytic activity by about 80% and 50% respectively, confirming the presence of zinc metalloproteolytic activity in the ECPs. Mass spectrometry analyses of crude ECPs identified an extracellular zinc metalloprotease encoded by a gene with an open reading frame of 1821 bp (606 aa). Consensus zinc-binding motifs specific to thermolysin family and some glycosylation and phosphorylation sites were located on the deduced protein sequence. Taken together, our results suggest that this (these) zinc metalloprotease(s) might contribute to the impairment of hemocyte immunological functions; however, their direct involvement in ECPs toxicity remains to be demonstrated. PMID:25252078

  16. A Bipartite Membrane-Binding Signal in the Human Immunodeficiency Virus Type 1 Matrix Protein Is Required for the Proteolytic Processing of Gag Precursors in a Cell Type-Dependent Manner

    PubMed Central

    Lee, Young-Min; Tian, Chun-Juan; Yu, Xiao-Fang

    1998-01-01

    It is unclear whether proteolytic processing of the human immunodeficiency virus type 1 (HIV-1) Gag protein is dependent on virus assembly at the plasma membrane. Mutations that prevent myristylation of HIV-1 Gag proteins have been shown to block virus assembly and release from the plasma membrane of COS cells but do not prevent processing of Gag proteins. In contrast, in HeLa cells similar mutations abolished processing of Gag proteins as well as virus production. We have now addressed this issue with CD4+ T cells, which are natural target cells of HIV-1. In these cells, myristylation of Gag proteins was required for proteolytic processing of Gag proteins and production of extracellular viral particles. This result was not due to a lack of expression of the viral protease in the form of a Gag-Pol precursor or a lack of interaction between unmyristylated Gag and Gag-Pol precursors. The processing defect of unmyristylated Gag was partially rescued ex vivo by coexpression with wild-type myristylated Gag proteins in HeLa cells. The cell type-dependent processing of HIV-1 Gag precursors was also observed when another part of the plasma membrane binding signal, a polybasic region in the matrix protein, was mutated. The processing of unmyristylated Gag precursors was inhibited in COS cells by HIV-1 protease inhibitors. Altogether, our findings demonstrate that the processing of HIV-1 Gag precursors in CD4+ T cells occurs normally at the plasma membrane during viral morphogenesis. The intracellular environment of COS cells presumably allows activation of the viral protease and proteolytic processing of HIV-1 Gag proteins in the absence of plasma membrane binding. PMID:9765451

  17. Single-cell analysis of dihydroartemisinin-induced apoptosis through reactive oxygen species-mediated caspase-8 activation and mitochondrial pathway in ASTC-a-1 cells using fluorescence imaging techniques

    NASA Astrophysics Data System (ADS)

    Lu, Ying-Ying; Chen, Tong-Sheng; Wang, Xiao-Ping; Li, Li

    2010-07-01

    Dihydroartemisinin (DHA), a front-line antimalarial herbal compound, has been shown to possess promising anticancer activity with low toxicity. We have previously reported that DHA induced caspase-3-dependent apoptosis in human lung adenocarcinoma cells. However, the cellular target and molecular mechanism of DHA-induced apoptosis is still poorly defined. We use confocal fluorescence microscopy imaging, fluorescence resonance energy transfer, and fluorescence recovery after photobleaching techniques to explore the roles of DHA-elicited reactive oxygen species (ROS) in the DHA-induced Bcl-2 family proteins activation, mitochondrial dysfunction, caspase cascade, and cell death. Cell Counting Kit-8 assay and flow cytometry analysis showed that DHA induced ROS-mediated apoptosis. Confocal imaging analysis in a single living cell and Western blot assay showed that DHA triggered ROS-dependent Bax translocation, mitochondrial membrane depolarization, alteration of mitochondrial morphology, cytochrome c release, caspase-9, caspase-8, and caspase-3 activation, indicating the coexistence of ROS-mediated mitochondrial and death receptor pathway. Collectively, our findings demonstrate for the first time that DHA induces cell apoptosis by triggering ROS-mediated caspase-8/Bid activation and the mitochondrial pathway, which provides some novel insights into the application of DHA as a potential anticancer drug and a new therapeutic strategy by targeting ROS signaling in lung adenocarcinoma therapy in the future.

  18. Proteolytic dissection of Zab, the Z-DNA-binding domain of human ADAR1

    NASA Technical Reports Server (NTRS)

    Schwartz, T.; Lowenhaupt, K.; Kim, Y. G.; Li, L.; Brown, B. A. 2nd; Herbert, A.; Rich, A.

    1999-01-01

    Zalpha is a peptide motif that binds to Z-DNA with high affinity. This motif binds to alternating dC-dG sequences stabilized in the Z-conformation by means of bromination or supercoiling, but not to B-DNA. Zalpha is part of the N-terminal region of double-stranded RNA adenosine deaminase (ADAR1), a candidate enzyme for nuclear pre-mRNA editing in mammals. Zalpha is conserved in ADAR1 from many species; in each case, there is a second similar motif, Zbeta, separated from Zalpha by a more divergent linker. To investigate the structure-function relationship of Zalpha, its domain structure was studied by limited proteolysis. Proteolytic profiles indicated that Zalpha is part of a domain, Zab, of 229 amino acids (residues 133-361 in human ADAR1). This domain contains both Zalpha and Zbeta as well as a tandem repeat of a 49-amino acid linker module. Prolonged proteolysis revealed a minimal core domain of 77 amino acids (positions 133-209), containing only Zalpha, which is sufficient to bind left-handed Z-DNA; however, the substrate binding is strikingly different from that of Zab. The second motif, Zbeta, retains its structural integrity only in the context of Zab and does not bind Z-DNA as a separate entity. These results suggest that Zalpha and Zbeta act as a single bipartite domain. In the presence of substrate DNA, Zab becomes more resistant to proteases, suggesting that it adopts a more rigid structure when bound to its substrate, possibly with conformational changes in parts of the protein.

  19. Lethal, neonatal ichthyosis with increased proteolytic processing of filaggrin in a mouse model of Netherton syndrome.

    PubMed

    Hewett, Duncan R; Simons, Alison L; Mangan, Niamh E; Jolin, Helen E; Green, Shelia M; Fallon, Padraic G; McKenzie, Andrew N J

    2005-01-15

    Netherton syndrome is an autosomal recessive multisystemic disorder characterized by congenital ichthyosiform erythroderma, hair shaft defects and atopy, caused by mutations within the human SPINK5 gene. To investigate the development of this disease, we have cloned mouse spink5 and created mice with a mutated premature stop codon at amino acid R820X, to produce an allele that closely mimics a point mutation (E827X) in human SPINK5. Newborn spink5(R820X/R820X) mice develop a lethal, severe ichthyosis with a loss of skin barrier function and dehydration, resulting in death within a few hours of birth, similar to that observed in patients with severe Netherton syndrome. Epidermal barrier function is compromised because of the stratum corneum becoming spontaneously detached in the newborn mice, and this is probably compounded by the reduced mechanical strength detected in the cornified envelopes. Biochemical analysis of skin from newborn wild-type and spink5(R820X/R820X) mice revealed a substantial increase in the proteolytic processing of profilaggrin into its constituent filaggrin monomers. Filaggrin functions to organize keratin filaments into highly ordered macrofibrils that crisscross the cornified cells of the stratum corneum imparting structural integrity, and defects in filaggrin processing occur in a number of forms of congenital ichthyosis. These data suggest that in the absence of the serine protease inhibitor spink5, there is an abnormal increase in the processing of profilaggrin, resulting in an overabundance of filaggrin monomers, and that this may play a direct role in the observed deficit in the adhesion of the stratum corneum and the severely compromised epidermal barrier function. PMID:15590704

  20. LEKTI proteolytic processing in human primary keratinocytes, tissue distribution and defective expression in Netherton syndrome.

    PubMed

    Bitoun, Emmanuelle; Micheloni, Alessia; Lamant, Laurence; Bonnart, Chrystelle; Tartaglia-Polcini, Alessandro; Cobbold, Christian; Al Saati, Talal; Mariotti, Feliciana; Mazereeuw-Hautier, Juliette; Boralevi, Franck; Hohl, Daniel; Harper, John; Bodemer, Christine; D'Alessio, Marina; Hovnanian, Alain

    2003-10-01

    SPINK5, encoding the putative multi-domain serine protease inhibitor LEKTI, was recently identified as the defective gene in the severe autosomal recessive ichthyosiform skin condition, Netherton syndrome (NS). Using monoclonal and polyclonal antibodies, we show that LEKTI is a marker of epithelial differentiation, strongly expressed in the granular and uppermost spinous layers of the epidermis, and in differentiated layers of stratified epithelia. LEKTI expression was also demonstrated in normal differentiated human primary keratinocytes (HK) through detection of a 145 kDa full-length protein and a shorter isoform of 125 kDa. Both proteins are N-glycosylated and rapidly processed in a post-endoplasmic reticulum compartment into at least three C-terminal fragments of 42, 65 and 68 kDa, also identified in conditioned media. Processing of the 145 and 125 kDa precursors was prevented in HK by treatment with a furin inhibitor. In addition, in vitro cleavage of the recombinant 145 kDa precursor by furin generated C-terminal fragments of 65 and 68 kDa, further supporting the involvement of furin in LEKTI processing. In contrast, LEKTI precursors and proteolytic fragments were not detected in differentiated HK from NS patients. Defective expression of LEKTI in skin sections was a constant feature in NS patients, whilst an extended reactivity pattern was obser