Removal of the Pro-Domain Does Not Affect the Conformation of the Procaspase-3 Dimer

C, Pop; Chen, Yun‐Ru; Smith, Brian; Bose, Kakoli; Bobay, Benjamin G.; Tripathy, Ashutosh; Franzen, Stefan; Ac, Clark

doi:10.1021/bi011037e

Cited by 72 publications

(120 citation statements)

References 53 publications

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“…At cytosolic concentration in human cells, the caspase 3 Pop et al, 2001) and 7 zymogens are already dimers (Boatright et al, 2003), but cleavage within their respective linker segments is required for activation (Chai et al, 2001b;Riedl et al, 2001a). The same reordering of catalytic and substrate binding residues occurs in caspase 7 as seen in caspase 9, so the fundamental mechanism of zymogen activation is equivalent.…”

Section: Execution Phase Activation: Caspases 3 Andmentioning

confidence: 99%

Caspase activation – stepping on the gas or releasing the brakes? Lessons from humans and flies

2004

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The central components of the execution phase of apoptosis in worms, flies, and humans are members of the caspase protease family. Work in Drosophila and mammalian systems has revealed a web of interactions that govern the activity of these proteases, and two fundamental control points have been identified. These are zymogen activation -the process that converts a latent caspase into its active form, and inhibition of the resulting active protease. In humans, the driving force for caspase activity is activation of the zymogens, but in Drosophila, a major thrust is derepression of caspase inhibitors. In this review, we consider evidence for these two distinct events in terms of the regulation of caspase activity. This sets the scene for therapy to reinstate the normal death mechanisms that have been overcome in a cancer cell's quest for immortality.

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Section: Execution Phase Activation: Caspases 3 Andmentioning

confidence: 99%

Caspase activation – stepping on the gas or releasing the brakes? Lessons from humans and flies

2004

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“…The products were analyzed by MALDI-TOF mass spectrometry and by SDS-PAGE using 4 to 25% or 10 to 25% polyacrylamide gels (7,19). The five N-terminal amino acids of each peptide were determined by Edman degradation (Protein Structure Core Facility, University of Nebraska,, Lincoln, NE).…”

Section: Trypsin and V8 Protease Digestionmentioning

confidence: 99%

Mutations in the Procaspase-3 Dimer Interface Affect the Activity of the Zymogen

et al. 2003

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The interface of the procaspase-3 dimer plays a critical role in zymogen maturation. We show that replacement of valine 266, the residue at the center of the procaspase-3 dimer interface, with glutamate resulted in an increase in enzyme activity of ~60-fold, representing a pseudoactivation of the procaspase. In contrast, substitution of V266 with histidine abolished the activity of the procaspase-3 as well as that of the mature caspase. While the mutations do not affect the dimeric properties of the procaspase, we show that the V266E mutation may affect the formation of a loop bundle that is important for stabilizing the active site. In contrast, the V266H mutation affects the positioning of loop L3, the loop that forms the bulk of the substrate binding pocket. In some cases, the amino acids affected by the mutations are >20 Å from the interface. Overall, the results demonstrate that the integrity of the dimer interface is important for maintaining the proper active site conformation.Programmed cell death is dependent on the maturation of the effector caspases from latent zymogens. While the procaspases are present at relatively high concentrations in the cell, it is not clear why they are enzymatically inactive. The mature caspase is a dimer of heterodimeric units containing a large subunit (17 kDa) and a small subunit (12 kDa). In the procaspase monomer, the structural units are organized as a short pro domain (28 amino acids), the large subunit, an intersubunit linker (25 amino acids), and the small subunit. Two monomers assemble into the procaspase homodimer that consists of a contiguous 12-stranded β-sheet core with several helices surrounding the β-sheet (see Figure 1). The procaspase is cleaved at D175, in the intersubunit linker, to remove the covalent connection between the subunits. The pro domain is then removed after cleavage at D9 and then at D28. Recently determined structures of procaspase-7 (1, 2) show that the core structural unit of the procaspase is comparable to that of the mature caspase (see Figure 1). The primary differences reside in five active site loops (for a review, see ref 3). Following maturation of procaspase-7, only the position of loop L1 (residues 52-67, caspase-3 numbering) remains unaltered. The data show that the procaspase is not enzymatically active because loop L3 (residues 198-213) is unraveled and positioned away from the active site, and the catalytic C163 is rotated away from solvent so that it cannot attack the substrate. The positioning of † This work was supported by a grant from the National Institutes of Health (GM065970) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptC163 is likely due to the covalent connection between loop L2 (residues 163-175) and the intersubunit linker (residues 176-192). Following cleavage at D175, loop L3 moves more than 10 Å toward the protein core to form the substrate binding pocket. Loop L2 moves toward L4, and the intersubunit linker, now called loop L2′, flips 180° to interact with loops L2 and L...

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“…Procaspase-8 contains N-terminal tandem DEDs, which provide interaction surfaces with adapter proteins and death receptors, 12,34 while procaspase-3 contains a short pro-domain (28 amino acids) in an extended structure. 24 The pro-domain of procaspase-3 has been shown to function as an intramolecular chaperone during subunit assembly, and it dramatically stabilizes the protease domains. 35 Likewise, the linkers (IL) that connect the large and small subunits in the protease domains have low sequence identity and appear to function differently in maintaining the inactive state.…”

Section: Discussionmentioning

confidence: 99%

“…For caspase-3, the residues in b8 present a flat, yet hydrophobic, face for each monomer, and the four inter-strand hydrogen bonds and a-helix 5 charge-charge interactions provide stabilizing interactions. Indeed, the K D for dimer dissociation of procaspase-3 was estimated to be <50 nM 24 whereas that for procaspase-8 was estimated to be 5 mM. 16,25 In the caspase-8 interface, one observes two inter-strand interactions between P466 and F468 in the center of the interface, where the sidechain of F468 stacks on the flat surface provided by P466' from the second monomer [ Fig.…”

Section: Introductionmentioning

confidence: 99%

Redesigning the procaspase‐8 dimer interface for improved dimerization

MacKenzie

Clark

2014

Protein Science

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Caspase-8 is a cysteine directed aspartate-specific protease that is activated at the cytosolic face of the cell membrane upon receptor ligation. A key step in the activation of caspase-8 depends on adaptor-induced dimerization of procaspase-8 monomers. Dimerization is followed by limited autoproteolysis within the intersubunit linker (IL), which separates the large and small subunits of the catalytic domain. Although cleavage of the IL stabilizes the dimer, the uncleaved procaspase-8 dimer is sufficiently active to initiate apoptosis, so dimerization of the zymogen is an important mechanism to control apoptosis. In contrast, the effector caspase-3 is a stable dimer under physiological conditions but exhibits little enzymatic activity. The catalytic domains of caspases are structurally similar, but it is not known why procaspase-8 is a monomer while procaspase-3 is a dimer. To define the role of the dimer interface in assembly and activation of procaspase-8, we generated mutants that mimic the dimer interface of effector caspases. We show that procaspase-8 with a mutated dimer interface more readily forms dimers. Time course studies of refolding also show that the mutations accelerate dimerization. Transfection of HEK293A cells with the procaspase-8 variants, however, did not result in a significant increase in apoptosis, indicating that other factors are required in vivo. Overall, we show that redesigning the interface of procaspase-8 to remove negative design elements results in increased dimerization and activity in vitro, but increased dimerization, by itself, is not sufficient for robust activation of apoptosis.

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Removal of the Pro-Domain Does Not Affect the Conformation of the Procaspase-3 Dimer

Cited by 72 publications

References 53 publications

Caspase activation – stepping on the gas or releasing the brakes? Lessons from humans and flies

Caspase activation – stepping on the gas or releasing the brakes? Lessons from humans and flies

Mutations in the Procaspase-3 Dimer Interface Affect the Activity of the Zymogen

Redesigning the procaspase‐8 dimer interface for improved dimerization

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