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Atsushi Takahashi - One of the best experts on this subject based on the ideXlab platform.

  • cleavage of lamin a by mch2 alpha but not cpp32 multiple interleukin 1 beta converting enzyme related proteases with distinct substrate recognition properties are active in apoptosis
    Proceedings of the National Academy of Sciences of the United States of America, 1996
    Co-Authors: Atsushi Takahashi, Emad S Alnemri, Yuri Lazebnik, Teresa Fernandesalnemri, Gerald Litwack, Robert D Moir, Robert D Goldman, Guy G Poirier
    Abstract:

    Although proteases related to the interleukin 1 beta-converting enzyme (ICE) are known to be essential for apoptotic execution, the number of enzymes involved, their substrate specificities, and their specific roles in the characteristic biochemical and morphological changes of apoptosis are currently unknown. These questions were addressed using cloned recombinant ICE-related proteases (IRPs) and a cell-free model system for apoptosis (S/M extracts). First, we compared the substrate specificities of two recombinant human IRPs, CPP32 and Mch2 alpha. Both enzymes cleaved poly-(ADP-ribose) polymerase, albeit with different efficiencies. Mch2 alpha also cleaved recombinant and nuclear lamin A at a conserved VEID decreases NG sequence located in the middle of the coiled-coil rod domain, producing a fragment that was indistinguishable from the lamin A fragment observed in S/M extracts and in apoptotic cells. In contrast, CPP32 did not cleave lamin A. The cleavage of lamin A by Mch2 alpha and by S/M extracts was inhibited by millimolar concentrations of Zn2+, which had a minimal effect on cleavage of poly (ADP-ribose) polymerase by CPP32 and by S/M extracts. We also found that N-(acetyltyrosinylvalinyl-N epsilon-biotinyllysyl)aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone, which derivatizes the larger subunit of active ICE, can Affinity Label up to five active IRPs in S/M extracts. Together, these observations indicate that the processing of nuclear proteins in apoptosis involves multiple IRPs having distinct preferences for their apoptosis-associated substrates.

  • cleavage of lamin a by mch2 alpha but not cpp32 multiple interleukin 1 beta converting enzyme related proteases with distinct substrate recognition properties are active in apoptosis
    Proceedings of the National Academy of Sciences of the United States of America, 1996
    Co-Authors: Atsushi Takahashi, Emad S Alnemri, Yuri Lazebnik, Teresa Fernandesalnemri, Gerald Litwack, Robert D Moir, Robert D Goldman, Guy G Poirier
    Abstract:

    Although proteases related to the interleukin 1 beta-converting enzyme (ICE) are known to be essential for apoptotic execution, the number of enzymes involved, their substrate specificities, and their specific roles in the characteristic biochemical and morphological changes of apoptosis are currently unknown. These questions were addressed using cloned recombinant ICE-related proteases (IRPs) and a cell-free model system for apoptosis (S/M extracts). First, we compared the substrate specificities of two recombinant human IRPs, CPP32 and Mch2 alpha. Both enzymes cleaved poly-(ADP-ribose) polymerase, albeit with different efficiencies. Mch2 alpha also cleaved recombinant and nuclear lamin A at a conserved VEID decreases NG sequence located in the middle of the coiled-coil rod domain, producing a fragment that was indistinguishable from the lamin A fragment observed in S/M extracts and in apoptotic cells. In contrast, CPP32 did not cleave lamin A. The cleavage of lamin A by Mch2 alpha and by S/M extracts was inhibited by millimolar concentrations of Zn2+, which had a minimal effect on cleavage of poly (ADP-ribose) polymerase by CPP32 and by S/M extracts. We also found that N-(acetyltyrosinylvalinyl-N epsilon-biotinyllysyl)aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone, which derivatizes the larger subunit of active ICE, can Affinity Label up to five active IRPs in S/M extracts. Together, these observations indicate that the processing of nuclear proteins in apoptosis involves multiple IRPs having distinct preferences for their apoptosis-associated substrates.

Guy G Poirier - One of the best experts on this subject based on the ideXlab platform.

  • cleavage of lamin a by mch2 alpha but not cpp32 multiple interleukin 1 beta converting enzyme related proteases with distinct substrate recognition properties are active in apoptosis
    Proceedings of the National Academy of Sciences of the United States of America, 1996
    Co-Authors: Atsushi Takahashi, Emad S Alnemri, Yuri Lazebnik, Teresa Fernandesalnemri, Gerald Litwack, Robert D Moir, Robert D Goldman, Guy G Poirier
    Abstract:

    Although proteases related to the interleukin 1 beta-converting enzyme (ICE) are known to be essential for apoptotic execution, the number of enzymes involved, their substrate specificities, and their specific roles in the characteristic biochemical and morphological changes of apoptosis are currently unknown. These questions were addressed using cloned recombinant ICE-related proteases (IRPs) and a cell-free model system for apoptosis (S/M extracts). First, we compared the substrate specificities of two recombinant human IRPs, CPP32 and Mch2 alpha. Both enzymes cleaved poly-(ADP-ribose) polymerase, albeit with different efficiencies. Mch2 alpha also cleaved recombinant and nuclear lamin A at a conserved VEID decreases NG sequence located in the middle of the coiled-coil rod domain, producing a fragment that was indistinguishable from the lamin A fragment observed in S/M extracts and in apoptotic cells. In contrast, CPP32 did not cleave lamin A. The cleavage of lamin A by Mch2 alpha and by S/M extracts was inhibited by millimolar concentrations of Zn2+, which had a minimal effect on cleavage of poly (ADP-ribose) polymerase by CPP32 and by S/M extracts. We also found that N-(acetyltyrosinylvalinyl-N epsilon-biotinyllysyl)aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone, which derivatizes the larger subunit of active ICE, can Affinity Label up to five active IRPs in S/M extracts. Together, these observations indicate that the processing of nuclear proteins in apoptosis involves multiple IRPs having distinct preferences for their apoptosis-associated substrates.

  • cleavage of lamin a by mch2 alpha but not cpp32 multiple interleukin 1 beta converting enzyme related proteases with distinct substrate recognition properties are active in apoptosis
    Proceedings of the National Academy of Sciences of the United States of America, 1996
    Co-Authors: Atsushi Takahashi, Emad S Alnemri, Yuri Lazebnik, Teresa Fernandesalnemri, Gerald Litwack, Robert D Moir, Robert D Goldman, Guy G Poirier
    Abstract:

    Although proteases related to the interleukin 1 beta-converting enzyme (ICE) are known to be essential for apoptotic execution, the number of enzymes involved, their substrate specificities, and their specific roles in the characteristic biochemical and morphological changes of apoptosis are currently unknown. These questions were addressed using cloned recombinant ICE-related proteases (IRPs) and a cell-free model system for apoptosis (S/M extracts). First, we compared the substrate specificities of two recombinant human IRPs, CPP32 and Mch2 alpha. Both enzymes cleaved poly-(ADP-ribose) polymerase, albeit with different efficiencies. Mch2 alpha also cleaved recombinant and nuclear lamin A at a conserved VEID decreases NG sequence located in the middle of the coiled-coil rod domain, producing a fragment that was indistinguishable from the lamin A fragment observed in S/M extracts and in apoptotic cells. In contrast, CPP32 did not cleave lamin A. The cleavage of lamin A by Mch2 alpha and by S/M extracts was inhibited by millimolar concentrations of Zn2+, which had a minimal effect on cleavage of poly (ADP-ribose) polymerase by CPP32 and by S/M extracts. We also found that N-(acetyltyrosinylvalinyl-N epsilon-biotinyllysyl)aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone, which derivatizes the larger subunit of active ICE, can Affinity Label up to five active IRPs in S/M extracts. Together, these observations indicate that the processing of nuclear proteins in apoptosis involves multiple IRPs having distinct preferences for their apoptosis-associated substrates.

Yuri Lazebnik - One of the best experts on this subject based on the ideXlab platform.

  • cleavage of lamin a by mch2 alpha but not cpp32 multiple interleukin 1 beta converting enzyme related proteases with distinct substrate recognition properties are active in apoptosis
    Proceedings of the National Academy of Sciences of the United States of America, 1996
    Co-Authors: Atsushi Takahashi, Emad S Alnemri, Yuri Lazebnik, Teresa Fernandesalnemri, Gerald Litwack, Robert D Moir, Robert D Goldman, Guy G Poirier
    Abstract:

    Although proteases related to the interleukin 1 beta-converting enzyme (ICE) are known to be essential for apoptotic execution, the number of enzymes involved, their substrate specificities, and their specific roles in the characteristic biochemical and morphological changes of apoptosis are currently unknown. These questions were addressed using cloned recombinant ICE-related proteases (IRPs) and a cell-free model system for apoptosis (S/M extracts). First, we compared the substrate specificities of two recombinant human IRPs, CPP32 and Mch2 alpha. Both enzymes cleaved poly-(ADP-ribose) polymerase, albeit with different efficiencies. Mch2 alpha also cleaved recombinant and nuclear lamin A at a conserved VEID decreases NG sequence located in the middle of the coiled-coil rod domain, producing a fragment that was indistinguishable from the lamin A fragment observed in S/M extracts and in apoptotic cells. In contrast, CPP32 did not cleave lamin A. The cleavage of lamin A by Mch2 alpha and by S/M extracts was inhibited by millimolar concentrations of Zn2+, which had a minimal effect on cleavage of poly (ADP-ribose) polymerase by CPP32 and by S/M extracts. We also found that N-(acetyltyrosinylvalinyl-N epsilon-biotinyllysyl)aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone, which derivatizes the larger subunit of active ICE, can Affinity Label up to five active IRPs in S/M extracts. Together, these observations indicate that the processing of nuclear proteins in apoptosis involves multiple IRPs having distinct preferences for their apoptosis-associated substrates.

  • cleavage of lamin a by mch2 alpha but not cpp32 multiple interleukin 1 beta converting enzyme related proteases with distinct substrate recognition properties are active in apoptosis
    Proceedings of the National Academy of Sciences of the United States of America, 1996
    Co-Authors: Atsushi Takahashi, Emad S Alnemri, Yuri Lazebnik, Teresa Fernandesalnemri, Gerald Litwack, Robert D Moir, Robert D Goldman, Guy G Poirier
    Abstract:

    Although proteases related to the interleukin 1 beta-converting enzyme (ICE) are known to be essential for apoptotic execution, the number of enzymes involved, their substrate specificities, and their specific roles in the characteristic biochemical and morphological changes of apoptosis are currently unknown. These questions were addressed using cloned recombinant ICE-related proteases (IRPs) and a cell-free model system for apoptosis (S/M extracts). First, we compared the substrate specificities of two recombinant human IRPs, CPP32 and Mch2 alpha. Both enzymes cleaved poly-(ADP-ribose) polymerase, albeit with different efficiencies. Mch2 alpha also cleaved recombinant and nuclear lamin A at a conserved VEID decreases NG sequence located in the middle of the coiled-coil rod domain, producing a fragment that was indistinguishable from the lamin A fragment observed in S/M extracts and in apoptotic cells. In contrast, CPP32 did not cleave lamin A. The cleavage of lamin A by Mch2 alpha and by S/M extracts was inhibited by millimolar concentrations of Zn2+, which had a minimal effect on cleavage of poly (ADP-ribose) polymerase by CPP32 and by S/M extracts. We also found that N-(acetyltyrosinylvalinyl-N epsilon-biotinyllysyl)aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone, which derivatizes the larger subunit of active ICE, can Affinity Label up to five active IRPs in S/M extracts. Together, these observations indicate that the processing of nuclear proteins in apoptosis involves multiple IRPs having distinct preferences for their apoptosis-associated substrates.

Roberta F. Colman - One of the best experts on this subject based on the ideXlab platform.

  • probing the active site of alpha class rat liver glutathione s transferases using Affinity Labeling by monobromobimane
    Protein Science, 2008
    Co-Authors: Longqin Hu, Barbara L Borleske, Roberta F. Colman
    Abstract:

    Monobromobimane (mBBr) is a substrate of both mu- and alpha-class rat liver glutathione S-transferases, with Km values of 0.63 microM and 4.9 microM for the mu-class isozymes 3-3 and 4-4, respectively, and 26 microM for the alpha-class isozymes 1-1 and 2-2. In the absence of substrate glutathione, mBBr acts as an Affinity Label of the 1-1 as well as mu-class isozymes, but not of the alpha-class 2-2 isozyme. Incubation of rat liver isozyme 1-1 with mBBr at pH 7.5 and 25 degrees C results in a time-dependent inactivation of the enzyme but at a slower (threefold) rate than for reactions with the mu-class isozyme 3-3 and 4-4. The rate of inactivation of 1-1 isozyme by mBBr is not decreased but, rather, is slightly enhanced by S-methyl glutathione. In contrast, 17 beta-estradiol-3,17-disulfate (500 microM) gives a 12.5-fold decrease in the observed rate constant of inactivation by 4 mM mBBr. When incubated for 60 min with 4 mM mBBr, the 1-1 isozyme loses 60% of its activity and incorporates 1.7 mol reagent/mol subunit. Peptide analysis after thermolysin digestion indicates that mBBr modification is equally distributed between two cysteine residues at positions 17 and 111. Modification at these two sites is reduced equally in the presence of the added protectant, 17 beta-estradiol-3,17-disulfate, suggesting that Cys 17 and Cys 111 reside within or near the enzyme's steroid binding sites. In contrast to the 1-1 isozyme, the other alpha-class isozyme (2-2) is not inactivated by mBBr at concentrations as high as 15 mM. The different reaction kinetics and modification sites by mBBr suggest that distinct binding site structures are responsible for the characteristic substrate specificities of glutathione S-transferase isozymes.

  • new insights from the structure function of the catalytic region of human platelet pde3a the role of the unique 44 amino acid insert
    Blood, 2005
    Co-Authors: Suhwi Hung, Roberta F. Colman, Wei Zhang, Jing Tao, Carolyn Kim, Rachel Noone, David Ezon, Robert W Colman
    Abstract:

    Human platelet PDE3A degrades cAMP, the major intracellular inhibitor of platelet function, and thus potentiates platelet activation. PDE3A is irreversibly inactivated by the Affinity Label Sp-cAMPS-BDB. The inactivation is prevented by Sp-cAMPS indicating that the Affinity Label is targeted at the cAMP binding site. We now use Sp-cAMPS-BDB with the aim of identifying nonconserved amino acids in substrate binding. After incubating Sp-cAMPS-BDB with PDE3A followed by reduction with [ 3 H]NaBH 4 , the incorporation was 1.1 mol/mol. HPLC analysis of the tryptic digest yielded a radioactive octapeptide T 806 YNVTDDK 813 in the 44-amino acid insert of PDE3A. Molecular modeling of PDE3A based on the PDE3B structure suggests the insert is a flexible loop. Incorporation of Sp-cAMPS-BDB indicates loop interaction with the substrate. Since Sp-cAMPS-BDB reacts with the nucleophilic residues, Y807, D811 and D812 were each mutated to alanine. Sp-cAMPS-BDB inactivates D811A and D812A but not Y807A, suggesting Y807 is the residue modified by Sp-cAMPS-BDB. Y807A affects the K m but not k cat , suggesting its involvement in cAMP binding. Kinetic analyses of 11 loop mutants reveal that H782A, T810A, Y814A and C816S each affects the k cat but not K m , indicating that catalysis is modulated. We conclude that binding of cAMP to the flexible loop of platelet PDE3A induces a conformational change which allows interaction with essential catalytic residues. These findings provide a new strategy for developing antiplatelet agents to treat patients with reocclusion of coronary arteries who are resistant to aspirin or whose chronic congestive heart failure prevents utilizing cilostazol.

  • activation of bovine liver glutamate dehydrogenase by covalent reaction of adenosine 5 o s 4 bromo 2 3 dioxobutyl thiophosphate with arginine 459 at an adp regulatory site
    Biochemistry, 1994
    Co-Authors: Kazimierz O Wrzeszczynski, Roberta F. Colman
    Abstract:

    : Bovine liver glutamate dehydrogenase is an allosteric enzyme which is activated by ADP. The Affinity Label adenosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)thiophosphate] (AMPSBDB), a new ADP analog featuring a reactive group at a position equivalent to that of the pyrophosphate, reacts with this glutamate dehydrogenase to yield enzyme containing about 0.9 mol/mol of enzyme subunit. The reaction results in a time-dependent irreversible activation of the enzyme. Glutamate dehydrogenase (8.9 microM subunit) modified with 10-60 microM AMPSBDB is about 3.2-fold more active than native enzyme. The modified enzyme is still inhibited by GTP and by high concentrations of NADH, but is no longer activated by ADP. The addition to the reaction mixture of (a) NADH or alpha-ketoglutarate; (b) GTP + NADH; or (c) alpha-ketoglutarate + NADH has little effect on the functional changes produced by AMPSBDB; whereas, the reaction is prevented by ADP. Purification of Labeled peptide from proteolytic and chemical digests of [2-3H]AMPSBDB-modified enzyme leads to identification of Arg459 as the target amino acid. We conclude that AMPSBDB functions as an ADP mimic covalently bound to Arg459 within the ADP activator site of the allosteric bovine liver glutamate dehydrogenase.

Bernard Henrissat - One of the best experts on this subject based on the ideXlab platform.

  • structure function analysis of a mixed linkage β glucanase xyloglucanase from the key ruminal bacteroidetes prevotella bryantii b14
    Journal of Biological Chemistry, 2016
    Co-Authors: Nicholas Mcgregor, Mariya Morar, Thomas Hauch Fenger, Nicolas Lenfant, Ekaterina Evdokimova, Peter J. Stogios, Xiaohui Xu, Bernard Henrissat
    Abstract:

    The recent classification of glycoside hydrolase family 5 (GH5) members into subfamilies enhances the prediction of substrate specificity by phylogenetic analysis. However, the small number of well characterized members is a current limitation to understanding the molecular basis of the diverse specificity observed across individual GH5 subfamilies. GH5 subfamily 4 (GH5_4) is one of the largest, with known activities comprising (carboxymethyl)cellulases, mixed-linkage endo-glucanases, and endo-xyloglucanases. Through detailed structure-function analysis, we have revisited the characterization of a classic GH5_4 carboxymethylcellulase, PbGH5A (also known as Orf4, carboxymethylcellulase, and Cel5A), from the symbiotic rumen Bacteroidetes Prevotella bryantii B14. We demonstrate that carboxymethylcellulose and phosphoric acid-swollen cellulose are in fact relatively poor substrates for PbGH5A, which instead exhibits clear primary specificity for the plant storage and cell wall polysaccharide, mixed-linkage β-glucan. Significant activity toward the plant cell wall polysaccharide xyloglucan was also observed. Determination of PbGH5A crystal structures in the apo-form and in complex with (xylo)glucan oligosaccharides and an active-site Affinity Label, together with detailed kinetic analysis using a variety of well defined oligosaccharide substrates, revealed the structural determinants of polysaccharide substrate specificity. In particular, this analysis highlighted the PbGH5A active-site motifs that engender predominant mixed-linkage endo-glucanase activity vis a vis predominant endo-xyloglucanases in GH5_4. However the detailed phylogenetic analysis of GH5_4 members did not delineate particular clades of enzymes sharing these sequence motifs; the phylogeny was instead dominated by bacterial taxonomy. Nonetheless, our results provide key enzyme functional and structural reference data for future bioinformatics analyses of (meta)genomes to elucidate the biology of complex gut ecosystems.