Tri-arginine exosite patch of caspase-6 recruits substrates for hydrolysis

Titus-McQuillan

et al. 2019

Preprint

Apoptotic caspases evolved with metazoans more than 950 million years ago (MYA), and a series of gene duplications resulted in two subfamilies consisting of initiator and effector caspases. The effector caspase genes (caspases-3, -6, and -7) were subsequently fixed into the Chordata phylum more than 650 MYA when the gene for a common ancestor (CA) duplicated, and the three effector caspases have persisted throughout mammalian evolution. All caspases require an aspartate residue at the P1 position of substrates, so each caspase evolved discrete cellular roles through changes in substrate recognition at the P4 position combined with allosteric regulation. We examined the evolution of substrate specificity in caspase-6, which prefers valine at the P4 residue, compared to caspases-3 and -7, which prefer aspartate, by reconstructing the CA of effector caspases (AncCP-Ef1) and the CA of caspase-6 (AncCP-6An). We show that AncCP-Ef1 is a promiscuous enzyme with little distinction between Asp, Val, or Leu at P4. The specificity of caspase-6 was defined early in its evolution, where AncCP-6An demonstrates preference for Val over Asp at P4. Structures of AncCP-Ef1 and of AncCP-6An show a network of charged amino acids near the S4 pocket that, when combined with repositioning a flexible active site loop, resulted in a more hydrophobic binding pocket in AncCP-6An. The ancestral protein reconstructions show that the caspasehemoglobinase fold has been conserved for over 650 million years and that only three substitutions in the scaffold are necessary to shift substrate selection toward Val over Asp.

Section: Introductionmentioning

confidence: 99%

Resurrection of ancestral effector caspases identifies novel networks for evolution of substrate specificity

Titus-McQuillan

et al. 2019

Preprint

“…As described previously ( 33 ), we quantified the rate of hydrolysis of the two procaspase substrates by assessing the disappearance of the full-length procaspases-3 and -6, both ∼32 kDa in size, and the appearance of the large (∼20 kDa) and small (∼10 kDa) subunits over the time course of the assay ( Fig. 5 , B and C ).…”

Section: Resultsmentioning

confidence: 99%

Caspases from scleractinian coral show unique regulatory features

Tung

Journal of Biological Chemistry

et al. 2020

Coral reefs are experiencing precipitous declines around the globe with coral diseases and temperature-induced bleaching being primary drivers of these declines. Regulation of apoptotic cell death is an important component in the coral stress response. Although cnidaria are known to 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 reef-building coral, and Porites astreoides, a disease-resistant reef-building coral. The caspases are predicted homologs of the human executioner caspases-3 and -7, but OfCasp3a (Orbicella faveolata caspase-3a) and PaCasp7a (Porites astreoides caspase-7a), which we show to be DxxDases, contain an amino-terminal caspase activation/recruitment domain (CARD) similar to human initiator/inflammatory caspases. OfCasp3b (Orbicella faveolata caspase-3b) and PaCasp3 (Porites astreoides caspase-3), which we show to be VxxDases, have short pro-domains, like human executioner caspases. Our biochemical analyses suggest a mechanism in coral which differs from that of humans, where the CARD-containing DxxDase is activated on death platforms but the protease does not directly activate the VxxDase. The first X-ray crystal structure of a coral caspase, of PaCasp7a determined at 1.57Å resolution, reveals a conserved fold and 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. These results suggest mechanisms for the evolution of substrate selection while maintaining common activation mechanisms of CARD-mediated dimerization.

“…Enzyme specificity of the four coral caspases was first examined by cleavage of human procaspases-3 and -6 in time-course assays, as described previously (34). The procaspase substrate was diluted to a final concentration of 5 µM in a buffer of 150 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1% sucrose, and 10 mM DTT at 37 °C.…”

Section: Whole-protein Cleavage Assaymentioning

confidence: 99%

“…The change in density for the procaspase substrate over time as a result of cleavage was quantified using Image lab software (Bio-Rad), and the data were plotted with Kaleidagraph. As described previously (34), the data were fit to an exponential decay to determine the CF50 (cleavage of 50% of protein substrate), and the CF50 was used to calculate the rate of hydrolysis (M -1 sec -1 ) using the equation k = ((−ln(P))/(E·t)). In this case, k is the rate of hydrolysis, P is the fraction cleaved (50%), E is the concentration at which CF50 is achieved (in molar), and t represents time (in seconds).…”

Section: Whole-protein Cleavage Assaymentioning

confidence: 99%

“…As described previously (34), we quantified the rate of hydrolysis of the two procaspase substrates by assessing the disappearance of the full-length procaspases-3 and -6, both ~32 kDa in size, and the appearance of the large (~20 kDa) and small (~10 kDa) subunits over the time course of the assay (Fig. 5B and Fig.…”

Section: Biochemical Characterization Of Coral Caspasesmentioning

confidence: 99%

See 1 more Smart Citation

Caspases from Scleractinian Coral Show Unique Regulatory Features

Tung

et al. 2020

Preprint

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.