Lengthening the Intersubunit Linker of Procaspase 3 Leads to Constitutive Activation

MacKenzie, S.H.; Schipper, J.L.; England, Erika J.; Thomas, Melvin E.; Blackburn, Kevin; Swartz, Paul; Clark, A. Clay

doi:10.1021/bi400793s

Cited by 16 publications

(16 citation statements)

References 44 publications

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“…Initial velocity of substrate cleavage was measured at 25°C in a buffer of 150 m M Tris‐HCl, pH 7.5, 50 m M NaCl, 0.1% CHAPS, 1% sucrose and 10 mM DTT in the presence of varying concentrations of Ac‐DEVD‐AFC or Ac‐VEID‐AMC substrates, as described previously for human CASP3 and caspase‐6 . The total reaction volume was 200 μL and the final enzyme concentration was 10 n M .…”

Section: Discussionmentioning

confidence: 99%

“…This Casp3a sequence differs from the reference sequence (NP_571952) by three residues at positions 57 (N‐>D), 180 (T‐>P), and 190 (E‐>V) and has been deposited in the GenBank database (accession KX084794). Escherichia coli BL21(DE3)pLysS cells were transformed with the plasmid, and Casp3a protein was expressed and purified as previously described for human CASP3 …”

Section: Methodsmentioning

confidence: 99%

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Phage display and structural studies reveal plasticity in substrate specificity of caspase‐3a from zebrafish

et al. 2016

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The regulation of caspase-3 enzyme activity is a vital process in cell fate decisions leading to cell differentiation and tissue development or to apoptosis. The zebrafish, Danio rerio, has become an increasingly popular animal model to study several human diseases because of their transparent embryos, short reproductive cycles, and ease of drug administration. While apoptosis is an evolutionarily conserved process in metazoans, little is known about caspases from zebrafish, particularly regarding substrate specificity and allosteric regulation compared to the human caspases. We cloned zebrafish caspase-3a (casp3a) and examined substrate specificity of the recombinant protein, Casp3a, compared to human caspase-3 (CASP3) by utilizing M13 bacteriophage substrate libraries that incorporated either random amino acids at P5-P1' or aspartate fixed at P1. The results show a preference for the tetrapeptide sequence DNLD for both enzymes, but the P4 position of zebrafish Casp3a also accommodates valine equally well. We determined the structure of zebrafish Casp3a to 2.28Å resolution by X-ray crystallography, and when combined with molecular dynamics simulations, the results suggest that a limited number of amino acid substitutions near the active site result in plasticity of the S4 sub-site by increasing flexibility of one active site loop and by affecting hydrogen-bonding with substrate. The data show that zebrafish Casp3a exhibits a broader substrate portfolio, suggesting overlap with the functions of caspase-6 in zebrafish development.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Phage display and structural studies reveal plasticity in substrate specificity of caspase‐3a from zebrafish

et al. 2016

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“…The codon optimized sequences of the four coral caspases, PaCasp3, PaCasp7a, OfCasp3a and OfCasp3b, were based on the sequences from previous transcriptomic data (20) and were cloned into pET11a vector (Genescript, USA). All proteins contained a C-terminal His6 tag and were expressed in E. coli BL21(DE3) pLysS cells and purified as previously described (26,27).…”

Section: Cloning Protein Expression and Protein Purificationmentioning

confidence: 99%

Caspases from Scleractinian Coral Show Unique Regulatory Features

Shrestha

Tung

Grinshpon

et al. 2020

Preprint

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Diseases affecting coral have led to massive decline and altered the community structure of reefs. In response to immune challenges, cnidaria activate apoptotic or autophagic pathways, and the particular pathway correlates with disease sensitivity (apoptosis) or resistance (autophagy).Although cnidaria contain complex apoptotic signaling pathways, similar to those in vertebrates, the mechanisms leading to cell death are largely unexplored. We identified and characterized two caspases each from Orbicella faveolata, a disease-sensitive stony coral, and Porites astreoides, a disease-resistant stony coral. The four caspases are predicted homologs of human caspases-3 and -7, but OfCasp3a and PaCasp7a contain an amino-terminal caspase activation and recruitment domain (CARD) similar to human initiator/inflammatory caspases. In contrast, OfCasp3b and PaCasp3 have short pro-domains, like human effector caspases. We show that OfCasp3a and PaCasp7a are DxxDases, like human caspases-3 and -7, while OfCasp3b and PaCasp3 are more similar to human caspase-6, with VxxDase activity. Our biochemical analyses suggest a mechanism in coral in which the CARD-containing DxxDase is activated on death platforms, but the protease does not directly activate the VxxDase. We also report the first X-ray crystal structure of a coral caspase, that of PaCasp7a determined at 1.57Å resolution. The structure reveals overall conservation of the caspase-hemoglobinase fold in coral as well as an N-terminal peptide bound near the active site that may serve as a regulatory exosite. The binding pocket has been observed in initiator caspases of other species, suggesting mechanisms for the evolution of substrate selection while maintaining common activation mechanisms of CARD-mediated dimerization.P. astreoides and O. faveolata contain VxxDases similar to HsCasp6. Our data show that the enzymes from both species have similar biochemical properties and are activated by similar mechanisms. Together, the data show that the role of the caspase cascade in disease resistance of P. astreoides or in disease sensitivity of O. faveolata may derive from differences in response mechanisms in the death receptor or the PIDDosome activation platforms, that is, signaling events upstream of the caspase cascade. Since the caspases in the two species exhibit similar biochemical properties and activation mechanisms, our data suggest that differences in the receptor-mediated activation of caspases as well as cross-talk between the autophagic and apoptotic pathways in the two coral species lead to the different physiological responses.

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“…It is possible that alternate cleavage sites by host proteases are the cause of this mature enzyme contamination. Indeed, proteolysis at D169 has been shown to occur when the normal cleavage site D175 is unavailable (MacKenzie et al, 2013). In our hands, such spurious activation in E. coli expression systems can be reduced by using shorter expression times, but it is impossible to eliminate completely.…”

Section: Small-molecule Activators Of Caspasesmentioning

confidence: 99%

Turning ON Caspases with Genetics and Small Molecules

Morgan

Julien

Unger

et al. 2014

Regulated Cell Death Part A: Apoptotic Mechanisms

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Caspases, aspartate-specific cysteine proteases, have fate-determining roles in many cellular processes including apoptosis, differentiation, neuronal remodeling, and inflammation (for review, see Yuan & Kroemer, 2010). There are a dozen caspases in humans alone, yet their individual contributions toward these phenotypes are not well understood. Thus, there has been considerable interest in activating individual caspases or using their activity to drive these processes in cells and animals. We envision that such experimental control of caspase activity can not only afford novel insights into fundamental biological problems but may also enable new models for disease and suggest possible routes to therapeutic intervention. In particular, localized, genetic, and small-molecule-controlled caspase activation has the potential to target the desired cell type in a tissue. Suppression of caspase activation is one of the hallmarks of cancer and thus there has been significant enthusiasm for generating selective small-molecule activators that could bypass upstream mutational events that prevent apoptosis. Here, we provide a practical guide that investigators have devised, using genetics or small molecules, to activate specific caspases in cells or animals. Additionally, we show genetically controlled activation of an executioner caspase to target the function of a defined group of neurons in the adult mammalian brain.

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Lengthening the Intersubunit Linker of Procaspase 3 Leads to Constitutive Activation

Cited by 16 publications

References 44 publications

Phage display and structural studies reveal plasticity in substrate specificity of caspase‐3a from zebrafish

Phage display and structural studies reveal plasticity in substrate specificity of caspase‐3a from zebrafish

Caspases from Scleractinian Coral Show Unique Regulatory Features

Turning ON Caspases with Genetics and Small Molecules

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