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

Agne Tubeleviciute-Aydin,Agne Tubeleviciute-Aydin,Andrea C Leblanc,Andrea C Leblanc,Libin Zhou,Libin Zhou,Sergey N Savinov,Sergey N Savinov,Ishankumar V Soni,Andrea C Leblanc,Jeanne A Hardy,Ishankumar V Soni,Gyanesh Sharma,Gyanesh Sharma

doi:10.1038/s41598-018-22283-z

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.

Highlights

Casp[6] is an attractive target for rational drug design against age-dependent cognitive impairment and Alzheimer Disease (AD)
We focus on two adjacent polymorphisms, Casp6-R65W (ExAC aggregated population allele frequency 0.0001159) and Casp6-G66R (Fig. 1a)
In the apo form, R65 and G66 are close to the active site but the two side-chain moieties are not interacting with other amino acid residues of Casp[6] and the side chain of R65 is pointing away from the Casp[6] active site catalytic dyad H121-C163

Summary

Introduction

Casp[6] is an attractive target for rational drug design against age-dependent cognitive impairment and AD. A recent novel null Casp[6] mouse possibly expressing a short catalytically inactive form of Casp[6], revealed increased cortical and striatal brain volumes and age-dependent learning deficits[26]. Casp[6] is expressed as an inactive dimeric zymogen (proCasp6) composed of a short pro-domain (Pro), a large subunit (LS) containing the active site cysteine-histidine catalytic dyad, an inter-subunit linker (L) and a small subunit (SS). The ligand-free Casp[6], differs from other caspases by the presence of 60’s and 90’s extended helices flanking the Casp[6] active site[4,28] Caspases cleave their substrates mostly at an aspartate, glutamate or phosphoserine residues[35]. Targeting allosteric sites is recognized as a more viable method for designing selective inhibitors for caspases, including Casp[64]. The existence of these Casp[6] variants suggests that full Casp[6] activity may be dispensable in humans

Methods

Results

Conclusion