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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 stress-induced 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-of-function 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-of-function character of these alleles. SIMILAR CONTENT BEING VIEWED BY OTHERS DNASE II MEDIATES A PARTHANATOS-LIKE DEVELOPMENTAL CELL DEATH PATHWAY IN

_DROSOPHILA_ PRIMORDIAL GERM CELLS Article Open access 16 April 2021 IONIZING RADIATION INDUCES CELLS WITH PAST CASPASE ACTIVITY THAT CONTRIBUTE TO THE ADULT ORGAN IN _DROSOPHILA_ AND SHOW

REDUCED LOSS OF HETEROZYGOSITY Article Open access 05 January 2024 AKT1 AND DCIZ1 PROMOTE CELL SURVIVAL FROM APOPTOTIC CASPASE ACTIVATION DURING REGENERATION AND ONCOGENIC OVERGROWTH Article

Open access 12 November 2020 MAIN Apoptosis 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 Caspase-3, -7 and -6.2, 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 imaginal discs.1, 2, 6 Caspase activation is tightly

regulated in surviving cells. Inhibitor of apoptosis proteins (IAPs) directly bind to and inhibit processed caspases.7, 8, 9 The best-characterized IAPs are mammalian XIAP and _Drosophila_

IAP1 (DIAP1).10, 11 In cells committed to apoptosis, IAP-mediated inhibition of caspases is counteracted by IAP antagonists. Specifically, the IAP antagonists encoded by _reaper_, _hid_ and

_grim_ (_RHG_)12, 13, 14, 15 in _Drosophila_ trigger proteolytic degradation of DIAP116, 17, 18, 19, 20 which releases caspases from DIAP1 inhibition and triggers apoptosis. The

overexpression of the _RHG_ genes in the fly eye using the eye-specific _GMR_ promoter causes an eye ablation phenotype due to massive apoptosis (see, for example, _GMR_-_hid_ in Figure

1a).12, 13, 14 In fact, mutants of _diap1_, _dronc_ and _drICE_ genes were isolated in genetic screens searching for modifiers of the eye ablation phenotypes caused by _reaper_ or _hid_

overexpression.21, 22, 23, 24, 25, 26, 27 Mammalian IAP antagonists are Smac/Diablo and HtrA2/Omi, which function similarly to the RHG proteins.28, 29, 30, 31 Both IAPs and IAP antagonists

are under tight control by various mechanisms to ensure proper regulation of caspase activity.1, 6, 11, 32 Given the pivotal roles of apoptosis in development and tissue homeostasis, it is

not surprising that deregulation of caspases has been implicated in various pathological conditions, including neurodegeneration, autoimmune diseases and cancers.33 Mutations in _Caspase-10_

and _-8_ are found in autoimmune lymphoproliferative syndrome (ALPS) and ALPS-related disorders.34 Mutations and polymorphisms of _Caspase-8_, _-9_, _-3_ and _-7_ have been implicated in

various cancers.35, 36 Although some of these mutations disrupt the apoptotic activity of the affected caspase in cell culture studies,37, 38, 39, 40, 41 detailed understanding of how these

mutations affect the function and expression of the caspases is scarce. It is thus of great biological and clinical significance to isolate more caspase mutants, especially the ones carrying

point mutations, and to analyze their behaviors _in vivo_. The _Drosophila_ model system provides a great venue to answer such inquiries, given the conservation of caspase genes and the

well-established genetic techniques to isolate and characterize mutations in genes of interest. DrICE and Dcp-1 are the effector caspase orthologs of mammalian Caspase-3 and Caspase-7 (ref.

42,43; reviewed in ref. 2, 6). Although Dcp-1 has a crucial role in nurse cell death during mid-oogenesis, DrICE is required for apoptosis in most developmental and irradiation-induced cell

death.25, 44, 45 Four _drICE_ mutants have been reported so far, only one of which, _drICE__17_, is caused by a missense mutation.25, 44, 46 _drICE__Δ1_, _drICE__Δ1E4_ and _drICE__Δ2C8_ were

generated through imprecise P-element excisions, through which the entire _drICE_ open-reading frame was removed to produce null alleles.44, 46 _drICE__17_ carries an N to Y mutation at

residue 116 and was isolated in an EMS mutagenesis screen as a recessive suppressor of the small eye phenotype induced by _GMR_-_hid_. This missense mutation leads to decreased protein

stability and behaves as a strong hypomorphic allele.25 These alleles have improved our understanding of the essential roles of _drICE_ in developmental, stress-induced and _RHG_-induced

cell death;25, 44, 46 however, they have not provided information on how various amino-acid substitutions affect the behavior of the caspase protein. In this study, we describe the isolation

and characterization of six new EMS-induced recessive loss-of-function alleles of _drICE_, all of which affect highly conserved amino-acid residues. Four of these alleles produce unstable

DrICE proteins. However, the remaining two alleles behave genetically differently. Although they were isolated as loss-of-function alleles homozygously, they surprisingly display

gain-of-function characteristics in the presence of a wild-type copy of _drICE_, that is, in a heterozygous condition. Interestingly, they only behave as gain-of-function mutants in the

presence of an apoptotic signal. Molecularly, the mutations in these alleles change conserved residues in close proximity to the catalytic Cys residue. We discuss models about how these

mutations cause the gain-of-function character of these alleles. RESULTS ISOLATION OF _DE NOVO DRICE_ MUTANT ALLELES Ectopic expression of _hid_ under the control of the eye-specific _GMR_

promoter causes an eye ablation phenotype owing to excessive cell death (Figure 1a).14 This eye ablation phenotype has been used in EMS mutagenesis screens to isolate mutants of genes

involved in apoptosis. To isolate additional _drICE_ mutant alleles to broaden our understanding of DrICE function in cell death, we performed an allele screen. The existing _drICE_ allele,

_drICE__17_, does not suppress the _GMR-hid_-induced eye ablation phenotype in a dominant or heterozygous manner (Figure 1b). Only homozygously (Figure 1c), or in trans to a _drICE_

deficiency (_drICE__Δ1_) (Figure 1d), can suppression of _GMR_-_hid_ be recorded. Therefore, to isolate new alleles of _drICE_, we took advantage of the fact that loss-of-heterozygosity of

_drICE__17_ causes strong suppression of _GMR_-_hid_. In an EMS mutagenesis screen, we screened 40 000 F1 progeny and recovered six suppressors, namely _L1, L2, C1, C2, S1_ and _S2_, which

suppressed _GMR_-_hid_ only in trans to _drICE__17_ (Figure 1e–j) and are therefore good candidates for new _drICE_ alleles. In addition, the six mutants are homozygously viable with wing

clearance defects (data not shown) as observed in _drICE__17_ and _drICE__Δ1_ alleles.25, 44 MOLECULAR CHARACTERIZATION OF THE NOVEL _DRICE_ ALLELES Through DNA sequencing, we identified in

each of these six alleles point mutations affecting amino acids highly conserved among effector caspases in various species (Figure 2a). _drICE__L1_ and _drICE__L2_ contain missense

mutations of amino acids located in the large subunit, changing Y86 to N and G94 to D, respectively. _drICE__S1_ and _drICE__S2_ bear nonsense mutations in the small subunit at positions 258

and 266, respectively, and produce carboxyl-end truncated _drICE_ proteins. _drICE__C1_ and _drICE__C2_ carry missense mutations of amino acids located near the catalytic cysteine, C211,

changing G213 to D and G219 to E, respectively. We refer to the alleles affecting the large subunit as class L alleles (_L1_ and _L2_), except the ones located close to the catalytic

cysteine residue, which we refer to as class C alleles (_C1_ and _C2_). Mutants affecting the small subunit are class S alleles (_S1_ and _S2_) (Figure 2b). DRICE PROTEIN LEVELS ARE MARKEDLY

DECREASED IN CLASS L AND CLASS S MUTANTS, BUT NOT IN CLASS C ALLELES To assess how these novel _drICE_ mutations affect DrICE function, we examined the stability of the mutant DrICE

proteins. Mosaic eye imaginal discs of the _drICE_ alleles from third instar larvae were labeled with an anti-DrICE polyclonal antibody.47 Levels of DrICE protein decreased markedly in

mutant clones of class L and class S alleles (Figure 3a’–e’), similar to _drICE__17_, which was previously shown to encode an unstable DrICE protein (ref. 25). These results suggest that

class L and class S alleles produce little or unstable DrICE protein. In contrast, class C alleles behave differently. Although mutant clones of _drICE__C1_ contain slightly decreased

protein levels, _drICE__C2_ produces normal DrICE protein levels comparable to wild-type tissues (Figure 3f’ and g’). To verify that the class C alleles indeed encode recessive

loss-of-function alleles, we examined if they affect normal developmental cell death during eye development. At 28 h after puparium formation (APF), a wave of apoptosis removes all excess

interommatidial cells.48, 49, 50 This developmental cell death is lost in mutant clones of both class C alleles (Figure 4a’ and b’), suggesting that class C alleles are recessive

loss-of-function mutants. Therefore, because these two alleles produce normal DrICE protein levels, they affect DrICE activity independently of protein stability. Given that they have

mutations in highly conserved residues close to the catalytic cysteine residue (Figure 2), it is likely that these mutants have reduced catalytic activity. CLASS C ALLELES AFFECT SUBSTRATE

BINDING From a structural perspective, it is quite clear why these mutations (G213D and G219E) lead to inactivation of DrICE when they are homozygous. In caspases generally, the

substrate-binding groove loops 2, 3 and 4 from one half of the tetramer interact with loop 2′ from the opposite half of the tetramer to form an ordered substrate-binding groove (Figure 5a).

Loops 2 and 2′ form a lock that holds loops 3 and 4 into the proper conformation. If the loop 2/loop 2′ interaction is lost, loops 3 and 4 become disordered and the enzyme is unable to bind

substrate (Figure 5c). Based on examination of the DrICE structure (3SIP),51 residues G213 and G219 are both contained within loop 2 of DrICE. Neither of these positions can accommodate any

residue larger than a glycine (Figure 5b) and the G213D and G219E mutations are predicted to inhibit critical interactions between loops 2 and 2’. G213 forms an exceptionally tight contact

with the backbone of F256 on loop 3, the loop that forms the base of the substrate-binding groove (Figure 5b). Substitution of G213 with aspartate disrupts the contact with F256 and prevents

the tight association of loops 2 and 2′, which supports substrate binding. G219, also on loop 2, interacts with the side chain of V241, which is contained on loop 2′ (Figure 5b).

Substitution of G219 with any rotomers of glutamate results in steric clash with adjacent residues. This steric clash would be sufficient to prevent loop ordering and substrate binding

(Figure 5c), explaining the loss of enzymatic activity of these two mutants in a homozygous condition. CLASS C _DRICE_ ALLELES DOMINANTLY ENHANCE _GMR-REAPER_ The new _drICE_ alleles were

recovered as recessive suppressors of _GMR_-_hid_ (Figure 1). They are also recessive suppressors of _GMR_-_reaper_ (Supplementary Figure S1). Surprisingly, however, in the course of this

analysis, we noted that the two class C alleles—when heterozygous in trans to a wild-type (_drICE_+) allele—dominantly enhanced _GMR_-_reaper_ (Figure 6b), whereas class L and class S

alleles weakly suppress it (Figure 6a; Supplementary Figure S2). Class C alleles also appear to act as dominant enhancers of _GMR_-_hid_ (Supplementary Figure S3). One possibility by which

class C alleles dominantly enhance _GMR_-_reaper_ is through increased caspase activity. To examine this possibility, we performed fluorometric caspase assays with head extracts from

_GMR-reaper_ animals heterozygous for various _drICE_ alleles. Consistently, although the loss-of-function alleles _drICE__L2_ and _drICE__S1_ have lost significant caspase activity, the

class C allele _drICE__C1_ displayed increased caspase activity compared with _GMR-reaper_ alone (Figure 6e). Furthermore, more TUNEL-positive signals are observed in _GMR-reaper_ eye

imaginal discs heterozygous for class C alleles compared with _GMR-reaper_ in wild-type or heterozygous class L background (Figure 6f–j). Therefore, the dominant enhancement of the eye

ablation phenotype of _GMR_-_reaper_ by class C alleles is indeed due to increased cell death. To further characterize the new _drICE_ alleles, we examined DIAP1 degradation triggered by

reaper.16, 17, 18, 19, 20 In _GMR-reaper_ eye imaginal discs, DIAP1 degradation is very prominent in cells immediately posterior to the column of R8 photoreceptor neurons, which are very

resistant to apoptosis52 and where DIAP1 is not degraded (Figure 7a and b; R8 photoreceptor columns are marked by arrows; the zones of DIAP1 degradation by asterisks (*)). Interestingly,

reduced DrICE activity in heterozygous loss-of-function alleles partially protects DIAP1 from reaper-induced degradation (Figure 7c and d), which is consistent with the suppression of

_GMR-reaper_ by these alleles (Figure 6a). In contrast, the gain-of-function alleles _drICE__C1_ and _drICE__C2_ fail to protect DIAP1 from reaper-induced degradation (asterisks in Figure 6e

and f). These findings suggest that the complex interaction between reaper, DIAP1 and DrICE is affected by the class C alleles. We also examined if the class C alleles behaved as

gain-of-function alleles in the absence of an apoptotic signal such as _GMR_-_reaper_, and thus may cause inappropriate cell loss using the developing retina as a model. At 42 h APF, the

retina forms a highly regular lattice with a constant number of cells48, 49, 50 (Supplementary Figure S4). Inappropriate cell loss results in disruption of lattice symmetry and is easy to

score. However, for both class C alleles, we did not detect any irregularity in the appearance of the lattice that may indicate inappropriate cell loss (Supplementary Figure S4). This

analysis suggests that the class C mutations do not cause premature and inappropriate activation of DrICE in the absence of an apoptotic signal. Taken together, the class C alleles exert a

very complex genetic behavior. Homozygously mutant _drICE__C1_ and _drICE__C2_ are recessive loss-of-function alleles and produce mutant DrICE proteins with impaired substrate binding.

However, in the presence of functional DrICE protein encoded from the _drICE_+ allele, they trigger more cell death than normal and enhance the _GMR-reaper_ eye ablation phenotype. Thus, in

a heterozygous condition they behave as gain-of-function alleles. DISCUSSION In this study, we report the isolation and characterization of six new mutants of the _Drosophila_ effector

caspase _drICE_, the ortholog of mammalian caspase-3. According to the types and locations of the mutations in these six new _drICE_ alleles, the expression levels of the mutant DrICE

proteins and the genetic interactions with _GMR-hid_ or _GMR-reaper,_ we have grouped these mutants into three classes. Class L and S alleles carry point mutations in the large and small

subunits, respectively (Figure 2b). In addition, according to this classification criterion, _drICE__17_(N116Y) also belongs to the class L group. Both class L and class S alleles affect the

stability of their DrICE protein products and hence have reduced caspase activity. Overall, they are recessive loss-of-function _drICE_ alleles. The class C alleles _drICE__C1_ and

_drICE__C2_ carry missense mutations in conserved amino acids (G213D and G219E) in close proximity of the catalytic cysteine (C211) residue. In contrast to class L and class S alleles, these

mutations do not markedly impact DrICE protein stability. Most importantly, although these alleles act as loss-of-function alleles homozygously, in the presence of a wild-type _drICE_+

allele (i.e., heterozygously), they can enhance cell death induced by _reaper_ expression, suggesting that they can function as gain-of-function alleles. However, our analysis suggests that

class C mutants only behave as gain-of-function alleles in an apoptotic background such as _GMR_-_reaper_, whereas they do not cause spontaneous activation of DrICE in the absence of

apoptotic signals. Therefore, they can only act as gain-of-function mutants after proteolytic processing. The underlying mechanisms of the apoptotic enhancement by heterozygous class C

alleles are less clear. In the few cases where certain mutants behave as recessive loss-of-function and dominant gain-of-function alleles, dimerization is the underlying cause of this

different genetic behavior. For example, certain mutations of the _Toll_ receptor are recessive loss-of-function alleles, but are gain-of-function owing to physical interaction with the

wild-type allele.53, 54 Given that two processed DrICE molecules form an enzymatically active tetramer with two catalytic sites,2, 3 one can speculate that class C mutant subunits exert a

dominant effect via interaction with a wild-type DrICE molecule. Statistically, only 50% of the DrICE tetramers in the cell are composed of a wild-type and a class C subunit (referred to as

wt/class C heterotetramer; Figure 5d). The remaining 50% are either wild-type (Figure 5a) or class C tetramers (Figure 5c), the latter of which has no enzymatic activity. Thus, the wt/class

C heterotetramers actually have more activity as the data imply because they also compensate for the loss of activity of the class C tetramers. How can we explain the gain-of-function

activity of the wt/class C heterotetramers? Because of the loss of loop organization and substrate binding in the class C subunit (Figure 5), it is unlikely that the presence of the

wild-type subunit in the wt/class C heterotetramer 'rescues' the defect in the class C subunit. It is more likely that an upstream regulatory mechanism is deregulated by the

wt/class C heterotetramer. There are several possibilities. For example, because IAPs require effector caspases for inhibitory activity,55, 56 it may be possible that a wt/class C

heterotetramer does not produce as much active DIAP1 compared with a wild-type tetramer. Specifically, the N terminus of DIAP1 is an intramolecular inhibitor of DIAP1, keeping DIAP1 in an

auto-inhibited conformation, unable to bind and inhibit DrICE.51, 57, 58, 59 After caspase cleavage at D20 (likely by DrICE), the inhibitory N terminus is removed and now activated DIAP1 can

bind to DrICE and inhibit it. Because DIAP1 is a substrate of DrICE before it becomes an inhibitor, the wt/class C heterotetramer would produce less-active DIAP1 and thus would be

less-efficiently inhibited. Another possible model includes cooperativity of the binding of DIAP1 to the two active sites of the DrICE tetramer. Because the inhibitory region of DIAP1 (pink

rod in Figure 5d) binds to the substrate-binding domain of DrICE present in the 3SIP structure,51 DIAP1 does not bind to the mutant catalytic site of the class C subunit. If there is

cooperativity of the binding of DIAP1 to the two catalytic sites of DrICE, the failure of DIAP1 to bind to the class C catalytic side may affect the binding of DIAP1 to the catalytic site of

the wild-type subunit. Thus, the catalytic site of the wild-type subunit would be free from IAP inhibition, which should lead to increased activity only in the heterozygous condition.

Finally, we also take into account that class C alleles are only dominantly active in the presence of an apoptotic signal such as reaper (Figure 6,Supplementary Figure S4). Reaper and DrICE

compete for binding to the BIR1 domain of DIAP1.47 If reaper is in excess, it triggers degradation of DIAP1 (Figure 7b) and thus apoptosis.16, 17, 18, 19, 20 Therefore, owing to the failure

of DIAP1 to bind to the class C subunit, the wt/class C heterotetramer may shift the equilibrium towards binding of DIAP1 to reaper such that DIAP1 is more efficiently degraded by reaper,

resulting in higher enzymatic activity of the wt/class C heterotetramer. Consistent with this notion, reduced DrICE activity in heterozygous loss-of-function alleles partially protects DIAP1

from reaper-induced degradation (Figure 7c and d), whereas the class C alleles of DrICE fail to protect DIAP1 from degradation (Figure 7e and f), suggestive of decreased binding of the

wt/class C heterotetramer to DIAP1. These models to explain the gain-of-function behaviors of the class C alleles are not mutually exclusive and other models may be possible, too. To our

knowledge, 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. Because the affected residues of the class C alleles are conserved in other caspases (Figure 2a), it would be

of great interest to generate and test such mutations in other caspases to examine if it is a universal phenomenon. In _Caenorhabditis elegans_, a large collection of _ced-3_ mutants has

been characterized, but no gain-of-function alleles have been reported.60 Interestingly, one _ced-3_ allele, _n2433_, substitutes G360 with S. This glycine residue corresponds to G213 in

DrICE, which is changed to D in _drICE__C1_ (Figure 2). However, in contrast to _drICE__C1_, _n2433_ behaves as a dominant negative.60 It is unclear, if these different genetic behaviors are

due to the different amino-acid substitutions (S in _n2433_ vs D in _drICE__C1_) or to intrinsic differences between Ced-3 and DrICE. In mice, the _Melody_ mutation in Caspase-3 substitutes

the catalytic C with a S residue. This mutation also behaves as a dominant negative in a heterozygous condition.61 The only mutation in Caspase-3 that confers constitutively active

properties to Caspase-3 is the V266E substitution, which was characterized by _in vitro_ mutagenesis.62, 63 This mutation increases Caspase-3 activity 60-fold. V266 is located in the dimer

interface of the small subunit and the V266E mutation promotes dimerization of Caspase-3 without proteolytic processing.63 V266 is not conserved in DrICE or other caspases (Figure 2), but

even if it was, it is unlikely that such a mutation can be recovered _in vivo_ as it will cause dominant lethality due to excessive apoptosis. The allele-specific enhancement of apoptotic

activity by caspase mutants is also of clinical significance. For example, it might be of interest to determine whether such dominant mutations of caspases have a role in the pathogenesis

and development of neurodegenerative disorders, for which the underlying mechanism is apoptotic neuron loss. Sequence analysis of caspase genes in these patients will help in answering this

possibility. Conversely, it would also be of interest to design and screen for drugs that would slightly twist the conformation of caspase tetramers to increase their enzymatic activity and

induce cell death in tumors. MATERIALS AND METHODS ISOLATION AND IDENTIFICATION OF _DRICE_ MUTANT ALLELES An isogenized _FRT82B_ stock was used for EMS mutagenesis and also used as a control

for genetic analyses. _FRT82B_ males were treated with 25 mM EMS in 5% sucrose solution for 24 h. After recovery for 3 h, they were crossed to _GMR-hid drICE__17_ females, and incubated at

25 °C. In all, 40 000 F1 progeny were screened for suppression of the small eye phenotype of _GMR-hid drICE__17_. Six mutants were identified as _de novo drICE_ alleles by genetic analysis

and DNA sequencing as described in the Results section. FLY STOCKS AND GENETICS The following mutant and transgenic fly stocks were used: drICEL1; drICEL2; drICEC1; drICEC2; drICES1; drICES2

(this study); drICE17 (ref. 25); drICEΔ1 (ref. 44); GMR-hid;14 CyO,2xGMR-reaper;12 ey-FLP, FRT82B ubi-GFP;64 GMR-hid ey-FLP, FRT82B ubi-GFP.25 GMR-hid drICE17 is a recombinant chromosome

carrying a GMR-hid transgene and drICE17. Genetic mosaics for immunohistochemical analysis were obtained by crossing the FRT82B drICE alleles with ey-FLP; FRT82B drICE-/FRT82B ubi-GFP. DNA

SEQUENCING Genomic DNA from homozygous mutant flies or trans-heterozygous over _drICE__Δ1_flies was isolated and PCR-amplified using _drICE_-specific primers. PCR fragments were sequenced by

Sanger sequencing. The single amino-acid code was used. IMMUNOHISTOCHEMISTRY TUNEL and immunohistochemistry were carried out as described.65 Anti-DrICE antibody (a kind gift of Pascal

Meier) was raised in guinea pig47 and used at a dilution of 1 : 200. Anti-Dlg antibody (a kind gift of Georg Halder) was used at a dilution of 1 : 2000. Anti-DIAP1 (SK14) antibody (a kind

gift of Pascal Meier) was raised in guinea pig and used at a concentration of 1 : 400. Cy3- and Cy-5 fluorescent-conjugated secondary antibodies were obtained from Jackson ImmunoResearch

(West Grove, PA, USA). In general, 10–20 eye imaginal discs were analyzed, unless stated otherwise. Fluorescent Images were captured using a Zeiss Axio Imager Z1 with ApoTome technology or

an Olympus Optical FV500 confocal microscope. QUANTIFICATION OF _GMR-REAPER_-INDUCED EXCESSIVE CELL DEATH All TUNEL-positive cells in _GMR-reaper_ larval eye discs in various genotypes were

counted to indicate how much cell death was induced. For each genotype, 15–17 representative eye discs were counted. Significance was calculated by using unpaired two-tailed Student’s

_t_-test with a 95% confidence interval. FLUOROMETRIC CASPASE ASSAYS Fluorometric caspase assays were performed as described with modifications.66 Adult heads were dissected on ice and lysed

with a pestle in caspase assay buffer (50 mM HEPES, pH 7.5; 100 mM NaCl; 1 mM EDTA; 0.1% CHAPS; 10% sucrose; 5 mMDTT; 0.5% TritonX‐100; 4% glycerol; 1 × protease inhibitor cocktail

(Promega, Madison, WI, USA)) and then sonicated once for 7 s. Protein concentrations of the lysates were determined by Bradford Protein Assay. For each genotype, 40 μg of total protein

lysates were used, respectively. Protein lysates and 100 μM of the fluorometric substrate DEVD-AMC (MP Biomedicals, Santa Ana, CA, USA) were combined in a 96-well plate on ice. Reaction

volume was brought to 100 μl with caspase assay buffer. Spectrophotometer was used to measure fluorescence (excitation 385 nm and emission 460 nm) at 15 min intervals for 2 h at 37 °C.

ABBREVIATIONS * ALPS: autoimmune lymphoproliferative syndrome * APF: after puparium formation * Ced-3: cell death defective 3 * CyO: curly of Oster * Dcp-1: death caspase 1 * DIAP1:

death-associated inhibitor of apoptosis 1 * DNA: desoxyribonucleic acid * drICE: death-related ICE (interleukin converting enzyme) * dronc: death regulator Nedd2-like caspase * EMS: ethyl

methanesulfonate * Ey: eyeless * Flp: flippase * FRT82B: flippase recombination target at 82B * GFP: green fluorescent protein * Gh: GMR-hid * Hid: head involution defective * GMR: glass

multimer reporter * HtrA2: high-temperature-required protein A2 * IAP: inhibitor of apoptosis protein * PCR: polymerase chain reaction * R8: photoreceptor 8 * RHG: reaper, hid, grim *

Smac/Diablo: second mitochondria-derived activator of caspases/direct IAP binding protein with low pI * TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling * Ubi: ubiquitous

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caspase-3 revealed by high resolution X-ray structure analysis. _J Mol Biol_ 2006; 359: 1378–1388. Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We are thankful

to Georg Halder (Katholieke Universiteit Leuven, Belgium), Bruce Hay (Caltech, USA), and Pascal Meier (Institute of Cancer Research, UK) for fly stocks and antibodies. This work was

supported by the National Institute of General Medical Science (NIGMS) to AB. AUTHOR INFORMATION Author notes * D Xu Present address: Current address: Shodair Children’s Hospital, Helena,

MT, USA., AUTHORS AND AFFILIATIONS * The University of Texas MD Anderson Cancer Center, Houston, TX, USA Y Wu, J Garnett, D Xu, E R Flores & A Bergmann * Department of Molecular, Cell

and Cancer Biology, University of Massachusetts Medical School, Worcester, MA, USA J L Lindblad, H E Kamber Kaya & A Bergmann * University of Massachusetts Amherst, Amherst, MA, USA Y

Zhao & J Hardy Authors * Y Wu View author publications You can also search for this author inPubMed Google Scholar * J L Lindblad View author publications You can also search for this

author inPubMed Google Scholar * J Garnett View author publications You can also search for this author inPubMed Google Scholar * H E Kamber Kaya View author publications You can also search

for this author inPubMed Google Scholar * D Xu View author publications You can also search for this author inPubMed Google Scholar * Y Zhao View author publications You can also search for

this author inPubMed Google Scholar * E R Flores View author publications You can also search for this author inPubMed Google Scholar * J Hardy View author publications You can also search

for this author inPubMed Google Scholar * A Bergmann View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to A Bergmann.

ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no conflict of interest. ADDITIONAL INFORMATION Edited by DL Vaux Supplementary Information accompanies this paper on Cell Death

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characterization of two gain-of-function alleles of the effector caspase DrICE in _Drosophila_. _Cell Death Differ_ 23, 723–732 (2016). https://doi.org/10.1038/cdd.2015.144 Download citation

* Received: 04 February 2013 * Revised: 14 September 2015 * Accepted: 29 September 2015 * Published: 06 November 2015 * Issue Date: April 2016 * DOI: https://doi.org/10.1038/cdd.2015.144

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