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H R Horvitz - One of the best experts on this subject based on the ideXlab platform.
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the caenorhabditis elegans cell death protein ced 3 is a cysteine Protease with substrate specificities similar to those of the human cpp32 Protease
Genes & Development, 1996Co-Authors: S Shaham, H R HorvitzAbstract:The Caenorhabditis elegans cell-death gene ced-3 encodes a protein similar to mammalian interleukin-l(3-converting enzyme (ICE), a cysteine Protease implicated in mammalian apoptosis. We show that the full-length CED-3 protein undergoes proteolytic activation to generate a CED-3 cysteine Protease and that CED-3 Protease activity is required for killing cells by programmed cell death in C. elegans. We developed an easy and general method for the purification of CED-3/ICE-Iike Proteases and used this method to facilitate a comparison of the substrate specificities of four different purified cysteine Proteases. We found that in its substrate preferences CED-3 was more similar to the mammalian CPP32 Protease than to mammalian ICE or NEDD2/ICH-1 Protease. Our results suggest that different mammalian CED-3/ICE-Iike Proteases may have distinct roles in mammalian apoptosis and that CPP32 is a candidate for being a mammalian functional equivalent of CED-3.
Vasanti V Deshpande - One of the best experts on this subject based on the ideXlab platform.
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a serine alkaline Protease from the fungus conidiobolus coronatus with a distinctly different structure than the serine Protease subtilisin carlsberg
Archives of Microbiology, 1996Co-Authors: Sangita Uday Phadtare, Vasanti V DeshpandeAbstract:In view of the functional similarities between subtilisin Carlsberg and the alkaline Protease fromConidiobolus coronatus, the biochemical and structural properties of the two enzymes were compared. In spite of their similar biochemical properties, e.g., pH optima, heat stability, molecular mass, pI, esterase activity, and inhibition by diisopropyl fluorophosphate and phenylmethlysulfonylfluoride, the Proteases were structurally dissimilar as revealed by (1) their amino acid compositions, (2) their inhibition by subtilisin inhibitor, (3) their immunological response to specific anti-Conidiobolus Protease antibody, and (4) their tryptic peptide maps. Our results demonstrate that although they are functionally analogous, theConidiobolus Protease is structurally distinct from subtilisin Carlsberg. TheConidiobolus Protease was also different from other bacterial and animal Proteases (e.g. pronase, Protease K, trypsin, and chymotrypsin) as evidenced by their lack of response to anti-Conidiobolus Protease antibody in double diffusion and in neutralization assays. TheConidiobolus serine Protease fails to obey the general rule that proteins with similar functions have similar primary sequences and, thus, are evolutionarily related. Our results strengthen the concept of convergent evolution for serine Proteases and provide basis for research in evolutionary relationships among fungal, bacterial, and animal Proteases.
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evidence for controlled autoproteolysis of alkaline Protease a mechanism for physiological regulation of conidial discharge in conidiobolus coronatus
FEBS Journal, 1992Co-Authors: Sangita U Phadatare, M C Srinivasan, Vasanti V DeshpandeAbstract:The alkaline serine Protease of Conidiobolus coronatus was shown to be involved in its conidial discharge [Phadatare, S., Srinivasan, M. C., Deshpande, M. (1989) Arch. Microbiol. 153, 47–49]. To understand the regulation of conidial discharge, the mechanism of control of Protease activity was investigated, which revealed the presence of two electrophoretically separable intracellular Proteases (Protease I and Protease II). The formation of smaller and less-active Protease II coincided with the decrease in conidial discharge. In order to trace the origin of Protease II, the corresponding purified extracellular enzymes were compared with respect to their biochemical, physicochemical and immunological properties. The biochemical properties, such as optimum pH and temperature, stability, sensitivity to metal ions and substrate specificity were closely similar for both Proteases. Amino acid analysis revealed that Protease II is completely similar to Protease I, though Protease I contains an additional portion which is not contained in Protease II. Western-blot ELISA, immunotitration and determination of antigenic valencies also revealed the structural similarity between the two Proteases. Purified Protease I showed partial degradation to Protease II in vitro, the process being sensitive to phenylmethylsulfonyl fluoride, indicating its proteolytic nature. These results suggest that the formation of a less-active Protease by autoproteolysis represents a novel means of physiological regulation of Protease activity, which in turn regulates the conidial discharge in C. coronatus.
Dominique Michaud - One of the best experts on this subject based on the ideXlab platform.
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a Protease activity depleted environment for heterologous proteins migrating towards the leaf cell apoplast
Plant Biotechnology Journal, 2012Co-Authors: Charles Goulet, Moustafa Khalf, Marcandre Daoust, Frank Sainsbury, Dominique MichaudAbstract:Recombinant proteins face major constraints along the plant cell secretory pathway, including proteolytic processing compromising their structural integrity. Here, we demonstrate the potential of Protease inhibitors as in situ stabilizing agents for recombinant proteins migrating towards the leaf apoplast. Genomic data for Arabidopsis, rice and Nicotiana spp. were assessed to determine the relative incidence of Protease families in the cell secretory pathway. Transient expression assays with the model platform Nicotiana benthamiana were then performed to test the efficiency of Protease inhibitors in stabilizing proteins targeted to the apoplast. Current genomic data suggest the occurrence of Proteases from several families along the secretory pathway, including A1 and A22 Asp Proteases; C1A and C13 Cys Proteases; and S1, S8 and S10 Ser Proteases. In vitro Protease assays confirmed the presence of various Proteases in N. benthamiana leaves, notably pointing to the deposition of A1- and S1-type activities preferentially in the apoplast. Accordingly, transient expression and secretion of the A1/S1 Protease inhibitor, tomato cathepsin D inhibitor (SlCDI), negatively altered A1 and S1 Protease activities in this cell compartment, while increasing the leaf apoplast protein content by ~45% and improving the accumulation of a murine diagnostic antibody, C5-1, co-secreted in the apoplast. SlCYS9, an inhibitor of C1A and C13 Cys Proteases, had no impact on the apoplast Proteases and protein content, but stabilized C5-1 in planta, presumably upstream in the secretory pathway. These data confirm, overall, the potential of Protease inhibitors for the in situ protection of recombinant proteins along the plant cell secretory pathway. © 2011 The Authors. Plant Biotechnology Journal
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A Protease activity–depleted environment for heterologous proteins migrating towards the leaf cell apoplast
Plant Biotechnology Journal, 2011Co-Authors: Charles Goulet, Moustafa Khalf, Marc-andre D'aoust, Frank Sainsbury, Dominique MichaudAbstract:Recombinant proteins face major constraints along the plant cell secretory pathway, including proteolytic processing compromising their structural integrity. Here, we demonstrate the potential of Protease inhibitors as in situ stabilizing agents for recombinant proteins migrating towards the leaf apoplast. Genomic data for Arabidopsis, rice and Nicotiana spp. were assessed to determine the relative incidence of Protease families in the cell secretory pathway. Transient expression assays with the model platform Nicotiana benthamiana were then performed to test the efficiency of Protease inhibitors in stabilizing proteins targeted to the apoplast. Current genomic data suggest the occurrence of Proteases from several families along the secretory pathway, including A1 and A22 Asp Proteases; C1A and C13 Cys Proteases; and S1, S8 and S10 Ser Proteases. In vitro Protease assays confirmed the presence of various Proteases in N. benthamiana leaves, notably pointing to the deposition of A1- and S1-type activities preferentially in the apoplast. Accordingly, transient expression and secretion of the A1/S1 Protease inhibitor, tomato cathepsin D inhibitor (SlCDI), negatively altered A1 and S1 Protease activities in this cell compartment, while increasing the leaf apoplast protein content by ~45% and improving the accumulation of a murine diagnostic antibody, C5-1, co-secreted in the apoplast. SlCYS9, an inhibitor of C1A and C13 Cys Proteases, had no impact on the apoplast Proteases and protein content, but stabilized C5-1 in planta, presumably upstream in the secretory pathway. These data confirm, overall, the potential of Protease inhibitors for the in situ protection of recombinant proteins along the plant cell secretory pathway. © 2011 The Authors. Plant Biotechnology Journal
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Gel electrophoresis of proteolytic enzymes
Analytica Chimica Acta, 1998Co-Authors: Dominique MichaudAbstract:This paper reviews the major procedures devised to efficiently detect and analyze proteolytic enzymes and their inhibitors after gel electrophoresis. After some considerations on the extraction of these proteins from biological samples and on the choice of appropriate substrates for the detection of Protease activities, the major analytical schemes developed to detect electrophoretically separated Proteases are described. Given the well-known importance of protein Protease inhibitors in the regulation of endogenous proteolytic enzymes and in host-pathogen and host-predator interactions, the detection of these proteins in crude extracts by the combined use of gel electrophoresis and specific Proteases is then considered. The use of gel electrophoresis in the study of Protease/Protease inhibitor complexes, useful in characterizing Protease mechanistic classes and in monitoring the activity of recombinant proteinaceous inhibitors designed to regulate Proteases in various biological systems, is finally considered. Copyright (C) 1998 Elsevier Science B.V.
Emad S Alnemri - One of the best experts on this subject based on the ideXlab platform.
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activation of the ced3 ice related Protease cpp32 in cerebellar granule neurons undergoing apoptosis but not necrosis
The Journal of Neuroscience, 1997Co-Authors: Robert C Armstrong, Gerald Litwack, Emad S Alnemri, Kim Hoang, Smita Gaur, Donald S Karanewsky, Lawrence C Fritz, Kevin J TomaselliAbstract:Neuronal apoptosis occurs during nervous system development and after pathological insults to the adult nervous system. Inhibition of CED3/ICE-related Proteases has been shown to inhibit neuronal apoptosis in vitro and in vivo , indicating a role for these cysteine Proteases in neuronal apoptosis. We have studied the activation of the CED3/ICE-related Protease CPP32 in two in vitro models of mouse cerebellar granule neuronal cell death: K + /serum deprivation-induced apoptosis and glutamate-induced necrosis. Pretreatment of granule neurons with a selective, irreversible inhibitor of CED3/ICE family Proteases, ZVAD-fluoromethylketone, specifically inhibited granule neuron apoptosis but not necrosis, indicating a selective role for CED3/ICE Proteases in granule neuron apoptosis. Extracts prepared from apoptotic, but not necrotic, granule neurons contained a Protease activity that cleaved the CPP32 substrate Ac-DEVD-aminomethylcoumarin. Induction of the Protease activity was prevented by inhibitors of RNA or protein synthesis or by the CED3/ICE Protease inhibitor. Affinity labeling of the Protease activity with an irreversible CED3/ICE Protease inhibitor, ZVK(biotin)D-fluoromethylketone, identified two putative Protease subunits, p20 and p18, that were present in apoptotic but not necrotic granule neuron extracts. Western blotting with antibodies to the C terminus of the large subunit of mouse CPP32 (anti-CPP32) identified p20 and p18 as processed subunits of the CPP32 proenzyme. Anti-CPP32 specifically inhibited the DEVD-amc cleaving activity, verifying the presence of active CPP32 Protease in the apoptotic granule neuron extracts. Western blotting demonstrated that the CPP32 proenzyme was expressed in granule neurons before induction of apoptosis. These results demonstrate that the CED3/ICE homolog CPP32 is processed and activated during cerebellar granule neuron apoptosis. CPP32 activation requires macromolecular synthesis and CED3/ICE Protease activity. The lack of CPP32 activation during granule neuron necrosis suggests that proteolytic processing and activation of CED3/ICE Proteases are specific biochemical markers of apoptosis.
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molecular ordering of the fas apoptotic pathway the fas apo 1 Protease mch5 is a crma inhibitable Protease that activates multiple ced 3 ice like cysteine Proteases
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Srinivasa M. Srinivasula, Gerald Litwack, Teresa Fernandesalnemri, Manzoor Ahmad, Emad S AlnemriAbstract:The Fas/APO-1-receptor associated cysteine Protease Mch5 (MACH/FLICE) is believed to be the enzyme responsible for activating a Protease cascade after Fas-receptor ligation, leading to cell death. The Fas-apoptotic pathway is potently inhibited by the cowpox serpin CrmA, suggesting that Mch5 could be the target of this serpin. Bacterial expression of proMch5 generated a mature enzyme composed of two subunits, which are derived from the precursor proenzyme by processing at Asp-227, Asp-233, Asp-391, and Asp-401. We demonstrate that recombinant Mch5 is able to process/activate all known ICE/Ced-3-like cysteine Proteases and is potently inhibited by CrmA. This contrasts with the observation that Mch4, the second FADD-related cysteine Protease that is also able to process/activate all known ICE/Ced-3-like cysteine Proteases, is poorly inhibited by CrmA. These data suggest that Mch5 is the most upstream Protease that receives the activation signal from the Fas-receptor to initiate the apoptotic Protease cascade that leads to activation of ICE-like Proteases (TX, ICE, and ICE-relIII), Ced-3-like Proteases (CPP32, Mch2, Mch3, Mch4, and Mch6), and the ICH-1 Protease. On the other hand, Mch4 could be a second upstream Protease that is responsible for activation of the same Protease cascade in CrmA-insensitive apoptotic pathways.
S Shaham - One of the best experts on this subject based on the ideXlab platform.
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the caenorhabditis elegans cell death protein ced 3 is a cysteine Protease with substrate specificities similar to those of the human cpp32 Protease
Genes & Development, 1996Co-Authors: S Shaham, H R HorvitzAbstract:The Caenorhabditis elegans cell-death gene ced-3 encodes a protein similar to mammalian interleukin-l(3-converting enzyme (ICE), a cysteine Protease implicated in mammalian apoptosis. We show that the full-length CED-3 protein undergoes proteolytic activation to generate a CED-3 cysteine Protease and that CED-3 Protease activity is required for killing cells by programmed cell death in C. elegans. We developed an easy and general method for the purification of CED-3/ICE-Iike Proteases and used this method to facilitate a comparison of the substrate specificities of four different purified cysteine Proteases. We found that in its substrate preferences CED-3 was more similar to the mammalian CPP32 Protease than to mammalian ICE or NEDD2/ICH-1 Protease. Our results suggest that different mammalian CED-3/ICE-Iike Proteases may have distinct roles in mammalian apoptosis and that CPP32 is a candidate for being a mammalian functional equivalent of CED-3.