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Protease

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H R Horvitz – One of the best experts on this subject based on the ideXlab platform.

  • 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, 1996
    Co-Authors: S Shaham, H R Horvitz
    Abstract:

    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.

  • a serine alkaline Protease from the fungus conidiobolus coronatus with a distinctly different structure than the serine Protease subtilisin carlsberg
    Archives of Microbiology, 1996
    Co-Authors: Sangita Uday Phadtare, Vasanti V Deshpande
    Abstract:

    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.

  • evidence for controlled autoproteolysis of alkaline Protease a mechanism for physiological regulation of conidial discharge in conidiobolus coronatus
    FEBS Journal, 1992
    Co-Authors: Sangita U Phadatare, M C Srinivasan, Vasanti V Deshpande
    Abstract:

    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.

  • a Protease activity depleted environment for heterologous proteins migrating towards the leaf cell apoplast
    Plant Biotechnology Journal, 2012
    Co-Authors: Charles Goulet, Moustafa Khalf, Frank Sainsbury, Marcandre Daoust, Dominique Michaud
    Abstract:

    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

  • A Protease activity–depleted environment for heterologous proteins migrating towards the leaf cell apoplast
    Plant Biotechnology Journal, 2011
    Co-Authors: Charles Goulet, Moustafa Khalf, Frank Sainsbury, Marc-andre D'aoust, Dominique Michaud
    Abstract:

    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

  • Gel electrophoresis of proteolytic enzymes
    Analytica Chimica Acta, 1998
    Co-Authors: Dominique Michaud
    Abstract:

    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.