A designed redox‐controlled caspase

Witkowski, Witold A.; Hardy, James L.

doi:10.1002/pro.673

Cited by 10 publications

(11 citation statements)

References 59 publications

(61 reference statements)

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“…To ensure that activity was monitored in a cleaved (active) form, each phosphomimetic was expressed from a constitutively two-chain construct in which the large and small subunits are independently expressed (Witkowski and Hardy, 2011). Neither S30E nor T173E had a significant effect on caspase-7 kinetics (Figure 1C).…”

Section: Resultsmentioning

confidence: 99%

Dual Site Phosphorylation of Caspase-7 by PAK2 Blocks Apoptotic Activity by Two Distinct Mechanisms

2017

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Caspases, the cysteine proteases that execute apoptosis, are tightly regulated via phosphorylation by a series of kinases. Although all apoptotic caspases work in concert to promote apoptosis, different kinases regulate individual caspases. Several sites of caspase-7 phosphorylation have been reported, but without knowing the molecular details, it has been impossible to exploit or control these complex interactions, which normally prevent unwanted proliferation. During dysregulation, PAK2 kinase plays an alternative anti-apoptotic role, phosphorylating caspase-7 and promoting unfettered cell growth and chemotherapeutic resistance. PAK2 phosphorylates caspase-7 at two sites, inhibiting activity using two different molecular mechanisms, before and during apoptosis. Phosphorylation of caspase-7 S30 allosterically obstructs its interaction with caspase-9, preventing intersubunit linker processing, slowing or preventing caspase-7 activation. S239 phosphorylation renders active caspase-7 incapable of binding substrate, blocking later events in apoptosis. Each of these mechanisms is novel, representing new opportunities for synergistic control of caspases and their counterpart kinases.

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Section: Resultsmentioning

confidence: 99%

Dual Site Phosphorylation of Caspase-7 by PAK2 Blocks Apoptotic Activity by Two Distinct Mechanisms

2017

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“…The wild-type caspase-7 used was a constitutively two-chain corrected version in pET23b that is designed to independently express the large and small subunits of caspase-7 with a His 6 tag at the C terminus (77). In both caspase-6 and caspase-7, the protein constructs were designed to exclude the prodomain (residues 1-23 in both caspase-6 and caspase-7) and linker (residues 180 -193 in caspase-6; residues 199 -206 in caspase-7).…”

Section: Methodsmentioning

confidence: 99%

Caspase-6 Undergoes a Distinct Helix-Strand Interconversion upon Substrate Binding

Dagbay¹,

Bolik-Coulon²,

Savinov

et al. 2017

Journal of Biological Chemistry

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Edited by Norma AllewellCaspases are cysteine aspartate proteases that are major players in key cellular processes, including apoptosis and inflammation. Specifically, caspase-6 has also been implicated in playing a unique and critical role in neurodegeneration; however, structural similarities between caspase-6 and other caspase active sites have hampered precise targeting of caspase-6. All caspases can exist in a canonical conformation, in which the substrate binds atop a ␤-strand platform in the 130's region. This caspase-6 region can also adopt a helical conformation that has not been seen in any other caspases. Understanding the dynamics and interconversion between the helical and strand conformations in caspase-6 is critical to fully assess its unique function and regulation. Here, hydrogen/deuterium exchange mass spectrometry indicated that caspase-6 is inherently and dramatically more conformationally dynamic than closely related caspase-7. In contrast to caspase-7, which rests constitutively in the strand conformation before and after substrate binding, the hydrogen/ deuterium exchange data in the L2 and 130's regions suggested that before substrate binding, caspase-6 exists in a dynamic equilibrium between the helix and strand conformations. Caspase-6 transitions exclusively to the canonical strand conformation only upon substrate binding. Glu-135, which showed noticeably different calculated pK a values in the helix and strand conformations, appears to play a key role in the interconversion between the helix and strand conformations. Because caspase-6 has roles in several neurodegenerative diseases, exploiting the unique structural features and conformational changes identified here may provide new avenues for regulating specific caspase-6 functions for therapeutic purposes.Caspases are cysteine proteases that recognize aspartatecontaining substrates and are major players in key cellular processes, including apoptosis and inflammation. Caspase active sites contain a Cys-His dyad required for cleavage of peptide bonds adjacent to aspartate residues in select protein substrates. There are two main classes of caspases, initiator and executioner caspases, classified based on their cellular function and domain organization. Initiator caspases (caspase-2, -8, and -9) function upstream in the apoptotic pathway and activate the downstream executioner caspases (caspase-3, -6, and -7) by proteolytic cleavage at an intersubunit linker between large and small subunits. Executioner caspases then cleave a select group of protein targets to promote apoptosis. Initiator caspases generally exist as monomers and are subsequently activated by dimerization mediated by interaction with a molecular platform (e.g.

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“…57 b as previously reported. 36 Errors on K M and k cat are the average of the uncertainty in the fits of weighted hyperbolas for substrate titrations performed on three separate days. N.D. is not detectable; <0.01.…”

Section: Figurementioning

confidence: 99%

“…For example, a BCR-ABL-activated casp-8 was developed to selectively eliminate leukemic cells, 33 small-molecule activatable “SNIPer” versions of casp-3, -6 and-7 were built to dissect their non-redundant proteolytic roles, 34 a constitutively active and uninhibitable version of casp-3 was developed 35 and casp-7 has been handcuffed by an introduced disulfide bond for reversible reactivation only under intracellular reducing conditions. 36 All of these re-engineering efforts have relied on rational design because the four-chain nature of active caspases and the resulting hypermobility of the substrate-binding groove prior to substrate binding challenges many of the traditional selection, screening and engineering approaches for altering specificity. To address these difficulties, we used our caged-green fluorescent protein (Caspase Activatable-GFP or CA-GFP) family of reporters 37,38 as the basis for a directed evolution selection method, which is compatible with challenging proteases.…”

Section: Introductionmentioning

confidence: 99%

Reprogramming Caspase-7 Specificity by Regio-Specific Mutations and Selection Provides Alternate Solutions for Substrate Recognition

Hill

MacPherson

et al. 2016

ACS Chem. Biol.

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The ability to routinely engineer protease specificity can allow us to better understand and modulate their biology for expanded therapeutic and industrial applications. Here we report a new approach based on a caged green fluorescent protein (CA-GFP) reporter that allows for flow-cytometry-based selection in bacteria or other cell types enabling selection of intracellular protease specificity, regardless of the compositional complexity of the protease. Here we apply this approach to introduce the specificity of caspase-6 into caspase-7, an intracellular cysteine protease important in cellular remodeling and cell death. We found that substitution of substrate-contacting residues from caspase-6 into caspase-7 was ineffective, yielding an inactive enzyme, whereas saturation mutagenesis at these positions and selection by directed evolution produced active caspases. The process produced a number of non-obvious mutations that enabled conversion of the caspase-7 specificity to match caspase-6. The structures of the evolved-specificity caspase-7 (esCasp-7) revealed alternate binding modes for the substrate, including reorganization of an active site loop. Profiling the entire human proteome of esCasp-7 by N-terminomics demonstrated that the global specificity toward natural protein substrates is remarkably similar to that of caspase-6. Because the esCasp-7 maintained the core of caspase-7, we were able to identify a caspase-6 substrate, lamin C, that we predict relies on an exosite for substrate recognition. These reprogrammed proteases may be the first tool built with the express intent of distinguishing exosite dependent or independent substrates. This approach to specificity reprogramming should also be generalizable across a wide range of proteases.

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A designed redox‐controlled caspase

Cited by 10 publications

References 59 publications

Dual Site Phosphorylation of Caspase-7 by PAK2 Blocks Apoptotic Activity by Two Distinct Mechanisms

Dual Site Phosphorylation of Caspase-7 by PAK2 Blocks Apoptotic Activity by Two Distinct Mechanisms

Caspase-6 Undergoes a Distinct Helix-Strand Interconversion upon Substrate Binding

Reprogramming Caspase-7 Specificity by Regio-Specific Mutations and Selection Provides Alternate Solutions for Substrate Recognition

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