skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: A designed redox-controlled caspase

Abstract

Caspases are a powerful class of cysteine proteases. Introduction of activated caspases in healthy or cancerous cells results in induction of apoptotic cell death. In this study, we have designed and characterized a version of caspase-7 that can be inactivated under oxidizing extracellular conditions and then reactivated under reducing intracellular conditions. This version of caspase-7 is allosterically inactivated when two of the substrate-binding loops are locked together via an engineered disulfide. When this disulfide is reduced, the protein regains its full function. The inactive loop-locked version of caspase-7 can be readily observed by immunoblotting and mass spectrometry. The reduced and reactivated form of the enzyme observed crystallographically is the first caspase-7 structure in which the substrate-binding groove is properly ordered even in the absence of an active-site ligand. In the reactivated structure, the catalytic-dyad cysteine-histidine are positioned 3.5 {angstrom} apart in an orientation that is capable of supporting catalysis. This redox-controlled version of caspase-7 is particularly well suited for targeted cell death in concert with redox-triggered delivery vehicles.

Authors:
;  [1]
  1. (UMASS, Amherst)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
National Institutes of Health (NIH)
OSTI Identifier:
1025047
Resource Type:
Journal Article
Resource Relation:
Journal Name: Protein Sci.; Journal Volume: 20; Journal Issue: (8) ; 08, 2011
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; APOPTOSIS; CATALYSIS; CELL KILLING; CYSTEINE; DISULFIDES; ENZYMES; INDUCTION; MASS SPECTROSCOPY; ORIENTATION; PROTEINS

Citation Formats

Witkowski, Witold A., and Hardy, Jeanne A.. A designed redox-controlled caspase. United States: N. p., 2011. Web. doi:10.1002/pro.673.
Witkowski, Witold A., & Hardy, Jeanne A.. A designed redox-controlled caspase. United States. doi:10.1002/pro.673.
Witkowski, Witold A., and Hardy, Jeanne A.. 2011. "A designed redox-controlled caspase". United States. doi:10.1002/pro.673.
@article{osti_1025047,
title = {A designed redox-controlled caspase},
author = {Witkowski, Witold A. and Hardy, Jeanne A.},
abstractNote = {Caspases are a powerful class of cysteine proteases. Introduction of activated caspases in healthy or cancerous cells results in induction of apoptotic cell death. In this study, we have designed and characterized a version of caspase-7 that can be inactivated under oxidizing extracellular conditions and then reactivated under reducing intracellular conditions. This version of caspase-7 is allosterically inactivated when two of the substrate-binding loops are locked together via an engineered disulfide. When this disulfide is reduced, the protein regains its full function. The inactive loop-locked version of caspase-7 can be readily observed by immunoblotting and mass spectrometry. The reduced and reactivated form of the enzyme observed crystallographically is the first caspase-7 structure in which the substrate-binding groove is properly ordered even in the absence of an active-site ligand. In the reactivated structure, the catalytic-dyad cysteine-histidine are positioned 3.5 {angstrom} apart in an orientation that is capable of supporting catalysis. This redox-controlled version of caspase-7 is particularly well suited for targeted cell death in concert with redox-triggered delivery vehicles.},
doi = {10.1002/pro.673},
journal = {Protein Sci.},
number = (8) ; 08, 2011,
volume = 20,
place = {United States},
year = 2011,
month = 9
}
  • Chronic exposure to arsenic in rats led to gradual accumulation of the toxicant in erythrocytes causing oxidative stress in these cells. 4-Hydroxynonenal (4-HNE), a major aldehyde product of lipid peroxidation, contributed significantly to the cytopathological events observed during oxidative stress in the erythrocytes of exposed rats. 4-HNE triggered death signal cascade that was initiated with the formation of HNE-protein adducts in cytosol. HNE-protein adduct formation resulted in depletion of cytosolic antioxidants followed by increased generation of ROS. Results showed accumulation of hydrogen peroxide (H{sub 2}O{sub 2}) from the early stages of arsenic exposure, while superoxide (O{sub 2}{sup c}entre dot{sup -})more » and hydroxyl radical ({sup c}entre dotOH) also contributed to the oxidative stress during longer period of exposure. Suppression of antioxidant system coupled with increased generation of ROS eventually led to activation of caspase 3 during arsenic exposure. Attenuation of HNE-mediated activation of caspase 3 in presence of N-acetylcysteine (NAC) indicated the involvement of GSH in the process. Prevention of HNE-mediated degradation of membrane proteins in presence of Z-DEVD-FMK identified caspase 3 as the principal mediator of HNE-induced cellular damage during arsenic exposure. Degradation of band 3 followed by its aggregation on the red cell surface promoted immunologic recognition of redistributed band 3 by autologous IgG with subsequent attachment of C3b. Finally, the formation of C3b-IgG-band 3 immune complex accelerated the elimination of affected cells from circulation and led to the decline of erythrocyte life span during chronic arsenic toxicity.« less
  • This paper discusses nitrogen-filled relief valves used in the All American Pipeline. This pipeline runs from California to west Texas, crossing mountains and valleys, through both temperature extremes, and across numerous geologic faults. These extremes result in large pressure gradients developing in the pipelines because of the temperature changes and the elevation changes involved. Pressure variations, caused by valves, pumping changes, and other factors, must be relieved quickly to avoid overpressure and shock waves that can be greatly amplified by the presence of fittings such as elbows and branches. Dan-Flo gas-loaded surge control valves were selected and installed at severalmore » locations along this system. These valves are a controlled relief valve that uses nitrogen gas as a part of their return spring force. The paper reviews their design and performance.« less
  • Cited by 3
  • Cavity-enhanced light scattering from an ultracold gas in an optical lattice constitutes a quantum measurement with a controllable form of the measurement backaction. Time-resolved counting of scattered photons alters the state of the atoms without particle loss implementing a quantum nondemolition measurement. The conditional dynamics is given by the interplay between photodetection events (quantum jumps) and no-count processes. The class of emerging atomic many-body states can be chosen via the optical geometry and light frequencies. Light detection along the angle of a diffraction maximum (Bragg angle) creates an atom-number-squeezed state, while light detection at diffraction minima leads to the macroscopicmore » superposition states (Schroedinger cat states) of different atom numbers in the cavity mode. A measurement of the cavity transmission intensity can lead to atom-number-squeezed or macroscopic superposition states depending on its outcome. We analyze the robustness of the superposition with respect to missed counts and find that a transmission measurement yields more robust and controllable superposition states than the ones obtained by scattering at a diffraction minimum.« less