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Papers by Joseph Krebs

Cancer Research

Recent evidence suggests that mammalian cysteine proteases related to Caenorhabditis elegans CED-... more Recent evidence suggests that mammalian cysteine proteases related to Caenorhabditis elegans CED-3 are key components of mammalian programmed cell death or apoptosis. We have shown recently that the CPP32 and Mch2 alpha cysteine proteases cleave the apoptotic markers poly(ADP-ribose) polymerase (PARP) and lamins, respectively. Here we report the cloning of a new Ced-3/interleukin 1 beta-converting enzyme-related gene, designated Mch3, that encodes a protein with the highest degree of homology to CPP32 compared to other family members. An alternatively spliced isoform, named Mch3 beta, was also identified. Bacterially expressed recombinant Mch3 has intrinsic autocatalytic/autoactivation activity. The specific activity of Mch3 alpha toward the peptide substrate DEVD-7-amino-4-methylcoumarin and PARP resembles that of CPP32. Like interleukin 1 beta-converting enzyme and CPP32, the active Mch3 alpha is made of two subunits derived from a precursor (proMch3 alpha). It was of interest tha...

Cancer research, Jan 15, 1995

Recent evidence suggests that mammalian cysteine proteases related to Caenorhabditis elegans CED-... more Recent evidence suggests that mammalian cysteine proteases related to Caenorhabditis elegans CED-3 are key components of mammalian programmed cell death or apoptosis. We have shown recently that the CPP32 and Mch2 alpha cysteine proteases cleave the apoptotic markers poly(ADP-ribose) polymerase (PARP) and lamins, respectively. Here we report the cloning of a new Ced-3/interleukin 1 beta-converting enzyme-related gene, designated Mch3, that encodes a protein with the highest degree of homology to CPP32 compared to other family members. An alternatively spliced isoform, named Mch3 beta, was also identified. Bacterially expressed recombinant Mch3 has intrinsic autocatalytic/autoactivation activity. The specific activity of Mch3 alpha toward the peptide substrate DEVD-7-amino-4-methylcoumarin and PARP resembles that of CPP32. Like interleukin 1 beta-converting enzyme and CPP32, the active Mch3 alpha is made of two subunits derived from a precursor (proMch3 alpha). It was of interest tha...

The Journal of Cell Biology, 1999

The mechanism by which membrane-bound Bcl-2 inhibits the activation of cytoplasmic procaspases is... more The mechanism by which membrane-bound Bcl-2 inhibits the activation of cytoplasmic procaspases is unknown. Here we characterize an intracellular, membrane-associated form of procaspase-3 whose activation is controlled by Bcl-2. Heavy membranes isolated from control cells contained a spontaneously activatable caspase-3 zymogen. In contrast, in Bcl-2 overexpressing cells, although the caspase-3 zymogen was still associated with heavy membranes, its spontaneous activation was blocked. However, Bcl-2 expression had little effect on the levels of cytoplasmic caspase activity in unstimulated cells. Furthermore, the membrane-associated caspase-3 differed from cytosolic caspase-3 in its responsiveness to activation by exogenous cytochrome c. Our results demonstrate that intracellular membranes can generate active caspase-3 by a Bcl-2–inhibitable mechanism, and that control of caspase activation in membranes is distinct from that observed in the cytoplasm. These data suggest that Bcl-2 may c...

Journal of Medicinal Chemistry, 2005

A series of oxamyl dipeptides were optimized for pan caspase inhibition, anti-apoptotic cellular ... more A series of oxamyl dipeptides were optimized for pan caspase inhibition, anti-apoptotic cellular activity and in vivo efficacy. This structure-activity relationship study focused on the P4 oxamides and warhead moieties. Primarily on the basis of in vitro data, inhibitors were selected for study in a murine model of R-Fas-induced liver injury. IDN-6556 (1) was further profiled in additional in vivo models and pharmacokinetic studies. This first-in-class caspase inhibitor is now the subject of two Phase II clinical trials, evaluating its safety and efficacy for use in liver disease.

Biochemistry, 1993

The functional importance and structural determinants of a conserved hydrophobic pocket in human ... more The functional importance and structural determinants of a conserved hydrophobic pocket in human carbonic anhydrase I1 (CA 11) were probed by preparing and characterizing 13 amino acid substitutions at Leu-198, situated at the mouth of the pocket. The pH dependence of the esterase activity reveals that activity decreases (up to 120-fold) as the amino acid size and charge at position 198 are varied while the pKa of the zinc-bound water molecule increases (up to 1 pH unit). Intriguingly, the pH dependence of the Leu-198-Glu substitution is parabolic (pKas = 6 and 9), consistent with introduction of a general basecatalyzed mechanism. Kinetic characterization of C02/HC03-interconversion catalyzed by four variants (Leu-l98+Ala, His, Arg, and Glu) reveals that increasing the size of the hydrophobic pocket (Ala) does not compromise catalysis (=3-fold decrease); however, substitution of charged (Arg and Glu) and larger (His) amino acids decreases kcat/& for C02 hydration substantially (17-fold, 19-fold, and 10-fold, respectively) but not completely. log kcat/& for C02 hydration, HC03-dehydration, and p-nitrophenyl acetate hydrolysis correlates with the hydrophobicity of the residue at 198, likely reflecting desolvation or electrostatic destabilization of the ground state. The X-ray crystal structures of the Leu-1984His, Glu, and Arg variants (Nair & Christianson, 1993) indicate that the His and Glu side chains are accommodated by minor structural reorganization leading to a wider mouth for the hydrophobic pocket while the Arg side chain blocks the pocket. Infrared spectroscopy of C02 bound to either wild-type CA I1 or the Leu-198-Arg variant indicates that the Arg substitution both decreases the affinity and alters the position of C02 binding, suggesting that the hydrophobic pocket forms the C02 binding site in CA 11. Finally, a 1.5-fold increase (Leu-198-Ala) and 12-fold decrease (Leu-198-Arg) in k,, for COZ hydration, indicative of the rate constant for intramolecular proton transfer from zinc-bound water to His-64, are likely mediated by changes in the active site solvent structure. Carbonic anhydrase is a zinc metalloenzyme which efficiently catalyzes the hydration of C02 to form bicarbonate and a proton. Catalysis of C02 hydration by one isozyme, human carbonic anhydrase I1 (CA 11,' EC 4.2.1. l), approaches the diffusion-control limit at lo8 M-' s-l and has a maximum turnover rate of more than lo6 per second [for recent reviews, see Silverman and Lindskog (1988) and Christianson (1 99 l)]. CA I1 also catalyzes the hydrolysis of aromatic esters and is potently inhibited by sulfonamide compounds and monovalent anions. A crystal structure of CA I1 has been solved (Liljas et al., 1972) and refined (Eriksson et al., 1986, 1988; Hikansson et al., 1992) to 1 S4-A resolution, providing tremendous insights into structure-function relationships within the enzyme. The zinc cofactor lies at the base of the conical active site cleft Supported by National Institutes of Health Grants GM40602 (C.A.F.) and GM40117 (R.A.D.). C.A.F. received an American Heart Association Established Investigator Award and a David and Lucile Packard Foundation Fellowship in Science and Engineering.

Biochemistry, 1998

The anti-apoptotic protein p35 from baculovirus is thought to prevent the suicidal response of in... more The anti-apoptotic protein p35 from baculovirus is thought to prevent the suicidal response of infected insect cells by inhibiting caspases. Ectopic expression of p35 in a number of transgenic animals or cell lines is also anti-apoptotic, giving rise to the hypothesis that the protein is a general inhibitor of caspases. We have verified this hypothesis by demonstrating that purified recombinant p35 inhibits human caspase-1,-3,-6,-7,-8, and-10 with k ass values from 1.2 × 10 3 to 7 × 10 5 (M-1 s-1), and with upper limits of K i values from 0.1 to 9 nM. Inhibition of 12 unrelated serine or cysteine proteases was insignificant, implying that p35 is a potent caspase-specific inhibitor. Mutation of the putative inhibitory loop to favor caspase-1 resulted in a substantial decline in caspase-3 inhibition, but minimal changes in caspase-1 inhibition. The interaction of p35 with caspase-3, as a model of the inhibitory mechanism, revealed classic slow-binding inhibition, with both active-sites of the caspase-3 dimer acting equally and independently. Inhibition resulted from complex formation between the enzyme and inhibitor, which could be visualized under nondenaturing conditions, but was dissociated by SDS to give p35 cleaved at Asp87, the P 1 residue of the inhibitor. Complex formation requires the substrate-binding cleft to be unoccupied. Taken together, these data revealed that p35 is an active-site-directed inhibitor highly adapted to inhibiting caspases.

Biochemistry, 1993

Substitution of cysteine for threonine-199, the amino acid which hydrogen bonds with zincbound hy... more Substitution of cysteine for threonine-199, the amino acid which hydrogen bonds with zincbound hydroxide in wild-type carbonic anhydrase I1 (CAII), leads to the formation of a new His3Cys zinc coordination polyhedron. The optical absorption spectrum of the Co2+-substituted threonine-199-cysteine (T199C) variant and the three-dimensional structure [Ippolito, J. A., & Christianson, D. W. (1993) Biochemistry (following paper in this issue)] indicate that the new thiolate side chain coordinates to the metal ion, displacing the metal-bound solvent molecule. The engineered thiolate ligand increases zinc binding (4-fold) and decreases catalytic activity substantially (=l 03-fold) but not completely. However, this residual activity is due to an active species containing a zinc-bound solvent ligand with the cysteine-199 side chain occupying an alternate conformation. The equilibrium between these conformers reflects the energetic balance between the formation of the zinc-thiolate bond and structural rearrangements in the Ser-197-Cys-206 loop necessary to achieve this metal coordination. This designed His3Cys metal polyhedron may mimic the zinc binding site in the matrix metalloproteinase prostromelysin. Zinc coordination polyhedra in proteins exhibit variations in ligand number, charge, structure, and amino acid composition depending upon the role of the metal ion (Christianson, 1991; Vallee & Auld, 1990). Utilizing these natural proteinzinc complexes as paradigms, transition metal sites have been designed de novo into a variety of protein scaffoldings including antibodies (

Cancer Research

Recent evidence suggests that mammalian cysteine proteases related to Caenorhabditis elegans CED-... more Recent evidence suggests that mammalian cysteine proteases related to Caenorhabditis elegans CED-3 are key components of mammalian programmed cell death or apoptosis. We have shown recently that the CPP32 and Mch2 alpha cysteine proteases cleave the apoptotic markers poly(ADP-ribose) polymerase (PARP) and lamins, respectively. Here we report the cloning of a new Ced-3/interleukin 1 beta-converting enzyme-related gene, designated Mch3, that encodes a protein with the highest degree of homology to CPP32 compared to other family members. An alternatively spliced isoform, named Mch3 beta, was also identified. Bacterially expressed recombinant Mch3 has intrinsic autocatalytic/autoactivation activity. The specific activity of Mch3 alpha toward the peptide substrate DEVD-7-amino-4-methylcoumarin and PARP resembles that of CPP32. Like interleukin 1 beta-converting enzyme and CPP32, the active Mch3 alpha is made of two subunits derived from a precursor (proMch3 alpha). It was of interest tha...

Cancer research, Jan 15, 1995

Recent evidence suggests that mammalian cysteine proteases related to Caenorhabditis elegans CED-... more Recent evidence suggests that mammalian cysteine proteases related to Caenorhabditis elegans CED-3 are key components of mammalian programmed cell death or apoptosis. We have shown recently that the CPP32 and Mch2 alpha cysteine proteases cleave the apoptotic markers poly(ADP-ribose) polymerase (PARP) and lamins, respectively. Here we report the cloning of a new Ced-3/interleukin 1 beta-converting enzyme-related gene, designated Mch3, that encodes a protein with the highest degree of homology to CPP32 compared to other family members. An alternatively spliced isoform, named Mch3 beta, was also identified. Bacterially expressed recombinant Mch3 has intrinsic autocatalytic/autoactivation activity. The specific activity of Mch3 alpha toward the peptide substrate DEVD-7-amino-4-methylcoumarin and PARP resembles that of CPP32. Like interleukin 1 beta-converting enzyme and CPP32, the active Mch3 alpha is made of two subunits derived from a precursor (proMch3 alpha). It was of interest tha...

The Journal of Cell Biology, 1999

The mechanism by which membrane-bound Bcl-2 inhibits the activation of cytoplasmic procaspases is... more The mechanism by which membrane-bound Bcl-2 inhibits the activation of cytoplasmic procaspases is unknown. Here we characterize an intracellular, membrane-associated form of procaspase-3 whose activation is controlled by Bcl-2. Heavy membranes isolated from control cells contained a spontaneously activatable caspase-3 zymogen. In contrast, in Bcl-2 overexpressing cells, although the caspase-3 zymogen was still associated with heavy membranes, its spontaneous activation was blocked. However, Bcl-2 expression had little effect on the levels of cytoplasmic caspase activity in unstimulated cells. Furthermore, the membrane-associated caspase-3 differed from cytosolic caspase-3 in its responsiveness to activation by exogenous cytochrome c. Our results demonstrate that intracellular membranes can generate active caspase-3 by a Bcl-2–inhibitable mechanism, and that control of caspase activation in membranes is distinct from that observed in the cytoplasm. These data suggest that Bcl-2 may c...

Journal of Medicinal Chemistry, 2005

A series of oxamyl dipeptides were optimized for pan caspase inhibition, anti-apoptotic cellular ... more A series of oxamyl dipeptides were optimized for pan caspase inhibition, anti-apoptotic cellular activity and in vivo efficacy. This structure-activity relationship study focused on the P4 oxamides and warhead moieties. Primarily on the basis of in vitro data, inhibitors were selected for study in a murine model of R-Fas-induced liver injury. IDN-6556 (1) was further profiled in additional in vivo models and pharmacokinetic studies. This first-in-class caspase inhibitor is now the subject of two Phase II clinical trials, evaluating its safety and efficacy for use in liver disease.

Biochemistry, 1993

The functional importance and structural determinants of a conserved hydrophobic pocket in human ... more The functional importance and structural determinants of a conserved hydrophobic pocket in human carbonic anhydrase I1 (CA 11) were probed by preparing and characterizing 13 amino acid substitutions at Leu-198, situated at the mouth of the pocket. The pH dependence of the esterase activity reveals that activity decreases (up to 120-fold) as the amino acid size and charge at position 198 are varied while the pKa of the zinc-bound water molecule increases (up to 1 pH unit). Intriguingly, the pH dependence of the Leu-198-Glu substitution is parabolic (pKas = 6 and 9), consistent with introduction of a general basecatalyzed mechanism. Kinetic characterization of C02/HC03-interconversion catalyzed by four variants (Leu-l98+Ala, His, Arg, and Glu) reveals that increasing the size of the hydrophobic pocket (Ala) does not compromise catalysis (=3-fold decrease); however, substitution of charged (Arg and Glu) and larger (His) amino acids decreases kcat/& for C02 hydration substantially (17-fold, 19-fold, and 10-fold, respectively) but not completely. log kcat/& for C02 hydration, HC03-dehydration, and p-nitrophenyl acetate hydrolysis correlates with the hydrophobicity of the residue at 198, likely reflecting desolvation or electrostatic destabilization of the ground state. The X-ray crystal structures of the Leu-1984His, Glu, and Arg variants (Nair & Christianson, 1993) indicate that the His and Glu side chains are accommodated by minor structural reorganization leading to a wider mouth for the hydrophobic pocket while the Arg side chain blocks the pocket. Infrared spectroscopy of C02 bound to either wild-type CA I1 or the Leu-198-Arg variant indicates that the Arg substitution both decreases the affinity and alters the position of C02 binding, suggesting that the hydrophobic pocket forms the C02 binding site in CA 11. Finally, a 1.5-fold increase (Leu-198-Ala) and 12-fold decrease (Leu-198-Arg) in k,, for COZ hydration, indicative of the rate constant for intramolecular proton transfer from zinc-bound water to His-64, are likely mediated by changes in the active site solvent structure. Carbonic anhydrase is a zinc metalloenzyme which efficiently catalyzes the hydration of C02 to form bicarbonate and a proton. Catalysis of C02 hydration by one isozyme, human carbonic anhydrase I1 (CA 11,' EC 4.2.1. l), approaches the diffusion-control limit at lo8 M-' s-l and has a maximum turnover rate of more than lo6 per second [for recent reviews, see Silverman and Lindskog (1988) and Christianson (1 99 l)]. CA I1 also catalyzes the hydrolysis of aromatic esters and is potently inhibited by sulfonamide compounds and monovalent anions. A crystal structure of CA I1 has been solved (Liljas et al., 1972) and refined (Eriksson et al., 1986, 1988; Hikansson et al., 1992) to 1 S4-A resolution, providing tremendous insights into structure-function relationships within the enzyme. The zinc cofactor lies at the base of the conical active site cleft Supported by National Institutes of Health Grants GM40602 (C.A.F.) and GM40117 (R.A.D.). C.A.F. received an American Heart Association Established Investigator Award and a David and Lucile Packard Foundation Fellowship in Science and Engineering.

Biochemistry, 1998

The anti-apoptotic protein p35 from baculovirus is thought to prevent the suicidal response of in... more The anti-apoptotic protein p35 from baculovirus is thought to prevent the suicidal response of infected insect cells by inhibiting caspases. Ectopic expression of p35 in a number of transgenic animals or cell lines is also anti-apoptotic, giving rise to the hypothesis that the protein is a general inhibitor of caspases. We have verified this hypothesis by demonstrating that purified recombinant p35 inhibits human caspase-1,-3,-6,-7,-8, and-10 with k ass values from 1.2 × 10 3 to 7 × 10 5 (M-1 s-1), and with upper limits of K i values from 0.1 to 9 nM. Inhibition of 12 unrelated serine or cysteine proteases was insignificant, implying that p35 is a potent caspase-specific inhibitor. Mutation of the putative inhibitory loop to favor caspase-1 resulted in a substantial decline in caspase-3 inhibition, but minimal changes in caspase-1 inhibition. The interaction of p35 with caspase-3, as a model of the inhibitory mechanism, revealed classic slow-binding inhibition, with both active-sites of the caspase-3 dimer acting equally and independently. Inhibition resulted from complex formation between the enzyme and inhibitor, which could be visualized under nondenaturing conditions, but was dissociated by SDS to give p35 cleaved at Asp87, the P 1 residue of the inhibitor. Complex formation requires the substrate-binding cleft to be unoccupied. Taken together, these data revealed that p35 is an active-site-directed inhibitor highly adapted to inhibiting caspases.

Biochemistry, 1993

Substitution of cysteine for threonine-199, the amino acid which hydrogen bonds with zincbound hy... more Substitution of cysteine for threonine-199, the amino acid which hydrogen bonds with zincbound hydroxide in wild-type carbonic anhydrase I1 (CAII), leads to the formation of a new His3Cys zinc coordination polyhedron. The optical absorption spectrum of the Co2+-substituted threonine-199-cysteine (T199C) variant and the three-dimensional structure [Ippolito, J. A., & Christianson, D. W. (1993) Biochemistry (following paper in this issue)] indicate that the new thiolate side chain coordinates to the metal ion, displacing the metal-bound solvent molecule. The engineered thiolate ligand increases zinc binding (4-fold) and decreases catalytic activity substantially (=l 03-fold) but not completely. However, this residual activity is due to an active species containing a zinc-bound solvent ligand with the cysteine-199 side chain occupying an alternate conformation. The equilibrium between these conformers reflects the energetic balance between the formation of the zinc-thiolate bond and structural rearrangements in the Ser-197-Cys-206 loop necessary to achieve this metal coordination. This designed His3Cys metal polyhedron may mimic the zinc binding site in the matrix metalloproteinase prostromelysin. Zinc coordination polyhedra in proteins exhibit variations in ligand number, charge, structure, and amino acid composition depending upon the role of the metal ion (Christianson, 1991; Vallee & Auld, 1990). Utilizing these natural proteinzinc complexes as paradigms, transition metal sites have been designed de novo into a variety of protein scaffoldings including antibodies (