Genetic characterization of two gain-of-function alleles of the effector caspase DrICE in Drosophila

Abstract

Caspases are the executioners of apoptosis. Although much is known about their physiological roles and structures, detailed analyses of missense mutations of caspases are lacking. As mutations within caspases are identified in various human diseases, the study of caspase mutants will help to elucidate how caspases interact with other components of the apoptosis pathway and how they may contribute to disease. DrICE is the major effector caspase in Drosophila required for developmental and stressinduced cell death. Here, we report the isolation and characterization of six de novo drICE mutants, all of which carry point mutations affecting amino acids conserved among caspases in various species. These six mutants behave as recessive loss-offunction mutants in a homozygous condition. Surprisingly, however, two of the newly isolated drICE alleles are gain-of-function mutants in a heterozygous condition, although they are loss-of-function mutants homozygously. Interestingly, they only behave as gain-of-function mutants in the presence of an apoptotic signal. These two alleles carry missense mutations affecting conserved amino acids in close proximity to the catalytic cysteine residue. This is the first time that viable gain-of-function alleles of caspases are described in any intact organism and provides a significant exception to the expectation that mutations of conserved amino acids always abolish the pro-apoptotic activity of caspases. We discuss models about how these mutations cause the gain-offunction character of these alleles. Cell Death and Differentiation (2016) 23, 723-732; doi:10.1038/cdd.2015.144; published online 6 November 2015Apoptosis is a major form of programmed cell death. 1 The core apoptotic machinery is evolutionary conserved with caspases as the fundamental components. [1][2][3] Caspases are specific cysteine proteases that are produced as inactive zymogens composed of an N-terminal pro-domain, a large subunit region with the catalytic cysteine residue, and a small subunit region at the carboxyl end. 2,4 Depending on their structures and functions, caspases are grouped into initiator and effector caspases. 2,3 Initiator caspases possess long pro-domains, which facilitate the recruitment of initiator caspases into cell death signaling complexes for activation. 2,3,5 Effector caspases are activated by initiator caspase complexes through proteolytic processing, cleaving off the pro-domain and separating the large and small subunits. The active effector caspase is a tetramer composed of two large and two small subunits and contains two catalytic sites. 2,3 Activated effector caspases cleave many protein targets to trigger the physiological and morphological changes characteristic of apoptosis. In mammals, apoptotic initiator caspases are Caspase-8, -9, -2 and -10, and effector caspases involved in apoptosis are 3 Of the seven Drosophila caspases, only the initiator caspase Dronc (Caspase-9-like) and the effector caspases DrICE and Dcp-1 (Caspase-3-like) have been implicated in apoptosis in imagina...

An inhibitory mono-ubiquitylation of the Drosophila initiator caspase Dronc functions in both apoptotic and non-apoptotic pathways

“…Caspase activity assays were performed as described [ 86 , 87 ]. Briefly, adult heads were lysed in caspase assay buffer (50 mM HEPES pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.1% CHAPS, 10% sucrose, 5 mM DTT, 0.5% TritonX-100, 4% glycerol and protease inhibitors).…”

A Review of Neuroprotective Effects and Mechanisms of Ginsenosides From Panax Ginseng in Treating Ischemic Stroke

“…Ginsenoside Rb1 is one of the main bioactive saponins in ginseng, which could alleviate cerebral ischemia injury via modulating apoptosis, autophagy, oxidative, inflammation, BBB permeability, and promoting neurogenesis ( Jiang et al, 2013 ; Luo et al, 2014 ; Chen et al, 2015 ; Cheng et al, 2019 ). Apoptotic caspases further classified as initiator caspases (Caspase-8, -9, -10), and effector caspases (Caspase-3, -6, -7) based on their functions ( Wu Y. et al, 2016 ). Ginsenoside Rb1 could inhibit apoptosis and attenuate damaged neurons by downregulation of the expression of caspase-3, caspase-9 ( Liu A. et al, 2018 ), nitric oxide, and superoxide ( Ke et al, 2014 ), and up-regulating the expression of the mitochondrion associated antiapoptotic factor Bcl-xL ( Zhang et al, 2006 ).…”