Rare human Caspase-6-R65W and Caspase-6-G66R variants identify a novel regulatory region of Caspase-6 activity

Abstract

The cysteine protease Caspase-6 (Casp6) is a potential therapeutic target of Alzheimer Disease (AD) and age-dependent cognitive impairment. To assess if Casp6 is essential to human health, we investigated the effect of CASP6 variants sequenced from healthy humans on Casp6 activity. Here, we report the effects of two rare Casp6 amino acid polymorphisms, R65W and G66R, on the catalytic function and structure of Casp6. The G66R substitution eliminated and R65W substitution significantly reduced Casp6 catalytic activity through impaired substrate binding. In contrast to wild-type Casp6, both Casp6 variants were unstable and inactive in transfected mammalian cells. In addition, Casp6-G66R acted as a dominant negative inhibitor of wild-type Casp6. The R65W and G66R substitutions caused perturbations in substrate recognition and active site organization as revealed by molecular dynamics simulations. Our results suggest that full Casp6 activity may not be essential for healthy humans and support the use of Casp6 inhibitors against Casp6-dependent neurodegeneration in age-dependent cognitive impairment and AD. Furthermore, this work illustrates that studying natural single amino acid polymorphisms of enzyme drug targets is a promising approach to uncover previously uncharacterized regulatory sites important for enzyme activity.

Rare CASP6N73T variant associated with hippocampal volume exhibits decreased proteolytic activity, synaptic transmission defect, and neurodegeneration

“…Casp6WT and Casp6N73T zymogens generated more LSL than LS (Fig. 2 b) indicating preferred self-cleavage at D193, as previously observed with Casp6WT 14 , 33 , 46 . The catalytically inactive Casp6D(23,179,193)A was not processed, as expected, and migrated as full-length (FL) Casp6 at 34 kDa.…”

Identification of Allosteric Inhibitors against Active Caspase-6

“…Aiming to identify novel allosteric sites in Casp6, we have investigated how human Casp6 rare non-synonymous missense SNPs resulting in substitutions away from the Casp6 active site, affect Casp6 catalytic efficiency and structure. In contrast to our previous study describing significant loss of activity in Casp6-R65W and Casp6-G66R rare SNP variants 51 , in the four A34E, E35K, A109T, and T182S remote SNPs investigated here, only the E35K slightly decreased Casp6 catalytic efficiency for a peptide substrate. The crystal structure of apo Casp6-E35K did not reveal hints that could have elucidated a potential mechanism responsible for reduced Casp6 activity.…”

“…In this study, we investigated how naturally occurring rare missense variants of human CASP6 affect Casp6 activity and structure. Following our previous work, which described rare natural Casp6-R65W and Casp6-G66R variants with significantly impaired activity 51 , we examined four additional missense rare variants with single amino acid substitutions located remotely from Casp6 active site: Casp6-A34E, Casp6-E35K, Casp6-A109T, and Casp6-T182S (Table S1, Fig. S1).…”

“…Plasmids encoding full-length Casp6-WT and variants, large and small subunits of Casp6-WT and variants, and Casp6-C163A were expressed in E. coli BL21(DE3)pLysS strain (Promega, Fitchburg, WI, USA) and purified 51 . Purity was evaluated by SDS-PAGE and Coomassie blue staining, and protein concentration was measured using Quick Start Bradford 1x Dye Reagent (Bio-Rad Laboratories).…”

“…Casp6 active sites were quantified using zVAD-FMK (N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)-fluoromethylketone, MP Biomedicals, Santa Ana, CA, USA) 51,64 . Casp6 activity was measured in Stennicke’s buffer (SB: 20 mM piperazine-N,N′-bis-(2-ethanesulfonic acid) pH 7.2, 100 mM NaCl, 10 mM DTT, 1 mM EDTA, 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonic acid, 10% sucrose) 65 using 40 µM Ac-VEID-AFC (Ac-Val-Glu-Ile-Asp-7-Amino-4-trifluoromethylcoumarin, Enzo Life Sciences, Farmingdale, NY, USA) 64 and 10-250 nM active site titrated Casp6.…”