Cleavage of DFNA5 by caspase-3 during apoptosis mediates progression to secondary necrotic/pyroptotic cell death.

TLDR: This work shows that caspase-3 cleaves the GSDMD-related protein DFNA5 after Asp270 to generate a necroticDFNA5-N fragment that targets the plasma membrane to induce secondary necrosis/pyroptosis, and provides a molecular mechanism forsecondary necrosis.

Abstract: Apoptosis is a genetically regulated cell suicide programme mediated by activation of the effector caspases 3, 6 and 7. If apoptotic cells are not scavenged, they progress to a lytic and inflammatory phase called secondary necrosis. The mechanism by which this occurs is unknown. Here we show that caspase-3 cleaves the GSDMD-related protein DFNA5 after Asp270 to generate a necrotic DFNA5-N fragment that targets the plasma membrane to induce secondary necrosis/pyroptosis. Cells that express DFNA5 progress to secondary necrosis, when stimulated with apoptotic triggers such as etoposide or vesicular stomatitis virus infection, but disassemble into small apoptotic bodies when DFNA5 is deleted. Our findings identify DFNA5 as a central molecule that regulates apoptotic cell disassembly and progression to secondary necrosis, and provide a molecular mechanism for secondary necrosis. Because DFNA5-induced secondary necrosis and GSDMD-induced pyroptosis are dependent on caspase activation, we propose that they are forms of programmed necrosis.

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Thomas Jefferson University Thomas Jefferson University
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Department of Biochemistry and Molecular
Biology Faculty Papers
Department of Biochemistry and Molecular
Biology
1-3-2017
Cleavage of DFNA5 by caspase-3 during apoptosis mediates Cleavage of DFNA5 by caspase-3 during apoptosis mediates
progression to secondary necrotic/pyroptotic cell death. progression to secondary necrotic/pyroptotic cell death.
Corey Rogers
Thomas Jefferson University
Teresa Fernandes-Alnemri
Thomas Jefferson University
Lindsey Mayes
Thomas Jefferson University
Diana Alnemri
The Pennsylvania State University
Gino Cingolani
Thomas Jefferson University
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Recommended Citation Recommended Citation
Rogers, Corey; Fernandes-Alnemri, Teresa; Mayes, Lindsey; Alnemri, Diana; Cingolani, Gino; and
Alnemri, Emad S., "Cleavage of DFNA5 by caspase-3 during apoptosis mediates progression to
secondary necrotic/pyroptotic cell death." (2017).
Department of Biochemistry and Molecular
Biology Faculty Papers.
Paper 111.
https://jdc.jefferson.edu/bmpfp/111
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Authors Authors
Corey Rogers, Teresa Fernandes-Alnemri, Lindsey Mayes, Diana Alnemri, Gino Cingolani, and Emad S.
Alnemri
This article is available at Jefferson Digital Commons: https://jdc.jefferson.edu/bmpfp/111

ARTICLE
Received 8 Jun 2016
| Accepted 4 Nov 2016 | Published 3 Jan 2017
Cleavage of DFNA5 by caspase-3 during
apoptosis mediates progression to secondary
necrotic/pyroptotic cell death
Corey Rogers
1
, Teresa Fernandes-Alnemri
1
, Lindsey Mayes
1
, Diana Alnemri
2
, Gino Cingolani
1
& Emad S. Alnemri
1
Apoptosis is a genetically regulated cell suicide programme mediated by activation of the
effector caspases 3, 6 and 7. If apoptotic cells are not scavenged, they progress to a lytic and
inflammatory phase called secondary necrosis. The mechanism by which this occurs is
unknown. Here we show that caspase-3 cleaves the GSDMD-related protein DFNA5 after
Asp270 to generate a necrotic DFNA5-N fragment that targets the plasma membrane to
induce secondary necrosis/pyroptosis. Cells that express DFNA5 progress to secondary
necrosis, when stimulated with apoptotic triggers such as etoposide or vesicular stomatitis
virus infection, but disassemble into small apoptotic bodies when DFNA5 is deleted. Our
findings identify DFNA5 as a central molecule that regulates apoptotic cell disassembly and
progression to secondary necrosis, and provide a molecular mechanism for secondary
necrosis. Because DFNA5-induced secondary necrosis and GSDMD-induced pyroptosis are
dependent on caspase activation, we propose that they are forms of programmed necrosis.
DOI: 10.1038/ncomms14128
OPEN
1
Department of Biochemistry and Molecular Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
2
Schreyer Honors College, Pennsylvania State University, 10 E College Ave, University Park, Pennsylvania 16802, USA. Correspondence and requests for
materials should be addressed to E.S.A. (email: emad.alnemri@jefferson.edu).
NATURE COMMUNICATIONS | 8:14128 | DOI: 10.1038/ncomms14128 | www.nature.com/naturecommunications 1

P
rogrammed cell death (PCD) pathways have important
physiological roles in growth, survival, homeostasis
and innate immunity of all multicellular organisms. Two
important, yet phenotypically distinct, forms of PCD include
apoptosis and programmed necrosis
1
. Although apoptosis
is immunologically ‘silent’, programmed necrosis is an
inflammatory form of PCD characterized by cellular swelling,
lysis and release of pro-inflammatory molecules
1
. Programmed
necrosis is mediated by two distinct signalling pathways; the
necroptotic pathway induces necroptosis and the pyroptotic
pathway induces pyroptosis
1
. Necroptosis is triggered by
activation of receptor-interacting protein kinase-3 (RIPK3),
which phosphorylates the pseudokinase MLKL, causing it to
translocate to the plasma membrane to induce cell permeabiliza-
tion
2
. Pyroptosis is triggered primarily by activation of the
inflammatory caspases, which include caspase-1 and caspase-11
(caspase-4/-5 in humans)
3,4
. Caspase-1 is activated by
multiprotein complexes assembled by several proteins such as
NLRP3, NLRC4, AIM2, pyrin and NLRP1, collectively referred to
as canonical inflammasomes (reviewed in
5
). By contrast, human
caspase-4 and -5, and their mouse ortholog caspase-11, are
activated within non-canonical inflammasome complexes by
directly binding to lipopolysaccharide from Gram-negative
bacteria
4
. Studies have demonstrated that on activation of
inflammatory caspases by the canonical and non-canonical
pathways these caspases cleave a cellular substrate called
gasdermin D (GSDMD) after Asp276 (refs 6–8), generating a
necrotic N-terminal fragment capable of inducing pyroptosis by
forming pores in the plasma membrane
9–12
.
In contrast to programmed necrosis, apoptosis is a
non-inflammatory form of PCD mediated by activation of the
apoptotic caspases and can occur either via an extrinsic or an
intrinsic pathway
13,14
. Although the extrinsic pathway is activated
by signalling through cell surface death receptors, the intrinsic
pathway is activated by mitochondrial damage. However, both
pathways converge on the activation of the executioner caspases
(caspase-3, 6 and 7), which target 4600 substrates to orchestrate
morphological changes associated with apoptosis
14
. At the
terminal stage of apoptosis, cells are phagocytosed in vivo by
scavenger cells, such as macrophages or neutrophils. However, if
these cells are not removed in a timely fashion, as is the case
in vitro, they progress to a final phase called secondary necrosis
characterized by cytoplasmic swelling and plasma membrane
damage, similar to the phenotype of cells undergoing pyroptosis
or necroptosis
15,16
. The mechanism of secondary necrosis
and whether it is mediated by substrates of apoptotic caspases
is not clear.
DFNA5 belongs to the same gasdermin superfamily, as
GSDMD and has been implicated in the induction of cell death
and as a putative tumour suppressor
17–19
. Mutations in intron 7
of the DFNA5 gene have been shown to cause sensorineural
hearing loss because of skipping of exon 8 at the pre-mRNA level
and the translation of a C-terminally truncated protein
20,21
.
Although the full-length product does not have cytotoxic
activity, the truncated form does
18,19
. In addition, promoter
hypermethylation leading to DFNA5 inactivation has been
detected in 52% of primary gastric cancers making it a putative
tumour suppressor and further suggesting a role in promoting
cell death
22
. Finally, expression of DFNA5 is induced by the
transcription factor p53 in response to etoposide, a potent
inducer of apoptosis
23
.
Because DFNA5 is related to GSDMD, and its C-terminal
truncation has been shown to cause cell death
18,19
,we
investigated whether it is cleaved by apoptotic caspases to
induce secondary necrosis. Here we show that DFNA5 is a
physiological substrate for caspase-3. Mechanistically, caspase-3
cleaves DFNA5 after Asp270 to generate a necrotic DFNA5-N
fragment that translocates to the plasma membrane to
permeabilize it and induce secondary necrosis/pyroptosis. In
293T cells that stably express DFNA5, activation of caspase-3 by
stimulation of the mitochondrial apoptotic pathway with Bax
overexpression, or infection by the apoptosis-inducing vesicular
stomatitis virus (VSV) or encephalomyocarditis virus (ECMV)
results in cleavage of DFNA5 and induction of secondary
necrosis. Similarly, in WT and caspase-1/caspase-11 (casp-1/
11)-deficient macrophages, infection with VSV or treatment with
etoposide results in cleavage of endogenous DFNA5 into the
necrotic N-terminal fragment and induction of secondary
necrosis. Deletion of DFNA5 in macrophages mostly inhibits
VSV-induced and etoposide-induced secondary necrosis.
Interestingly, unlike WT cells, DFNA5-deficient cells do not
swell, but extensively disassemble into small apoptotic bodies.
Combined, our results indicate that DFNA5 regulates disassembly
and progression of apoptotic cells to secondary necrosis on
cleavage by caspase-3.
Results
DFNA5 is specifically cleaved by caspase-3. Our studies in
VSV-infected immortalized bone marrow-derived macrophages
(BMDMs) from casp-1/11-deficient mice revealed that these
macrophages undergo a caspase-dependent necrotic form of cell
death resembling pyroptosis. VSV-infected casp-1/11-deficient
macrophages released high amount of LDH in the culture
supernatants and showed microscopic features of plasma mem-
brane swelling characteristic of necrosis/pyroptosis (Supplemen-
tary Fig. 1). Pharmacological inhibition of the necroptotic path-
way with the RIPK3 inhibitor GSK’872 alone did not block this
cell death. However, combined treatment with GSK’872 and the
pancaspase-inhibitor zVAD-fmk completely blocked cell death,
indicating that this VSV-induced necrotic-like cell death is not
mediated by necroptosis but is mediated by caspases other than
caspase-1 and caspases-11. The observed high LDH release in the
presence of zVAD-fmk is caused by zVAD-fmk-induced
necroptosis, because inhibition of caspases leads to activation of
the RIPK3-MLKL necroptotic cell death pathway in macro-
phages
24
. Since casp-1/11 dKO macrophages lack inflammatory
caspases, which induce pyroptosis by cleaving GSDMD (refs 6–8),
these results suggest that the observed VSV-induced necrosis in
these cells is likely the result of non-inflammatory caspase activity
on substrates other than GSDMD. To test this hypothesis we
investigated whether non-inflammatory caspases can cleave
other GSDMD-related family members to induce necrosis-like
phenotype.
DFNA5, a GSDMD-related family member, shares only B28%
identity with GSDMD within the region corresponding to the
pyroptotic GSDMD N-terminal domain (Supplementary Fig. 2).
Nevertheless, genetic mutations within intron 7 of human
DFNA5 that cause skipping of exon 8 and truncation of the
C-terminus of DFNA5 at residue 315 leads to hearing loss
21
,
suggesting that the N-terminal domain of DFNA5, like GSDMD
N-terminal domain, may possess cell-death inducing activity.
As a first step to investigate whether DFNA5 is an effector
of necrotic-like cell death downstream of caspase activation,
we tested whether DFNA5 is a target for caspases. Incubation of
N-terminal T7 tagged recombinant DFNA5 with caspase-3,
but not with caspase-1, resulted in the generation of B35 kDa
N-terminal fragment similar in size to the caspase-1 generated
GSDMD N-terminal domain (Fig. 1a). Among human caspases
only caspase-3 was able to efficiently cleave DFNA5
(Supplementary Fig. 3a). Notably, caspase-3 was also capable of
cleaving GSDMD but resulted in the generation of a 13 kDa
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14128
2 NATURE COMMUNICATIONS | 8:14128 | DOI: 10.1038/ncomms14128 | www.nature.com/naturecommunications

GSDMD N-terminal fragment. This result is consistent with
a previous report
25
, which showed that GSDMD is cleaved at
Asp87 during apoptosis (http://wellslab.ucsf.edu/cgibin/retrieve_
1.0.cgi?degrabase_type=both&p1_type=all&substrate=gsdmd).
This residue is part of a consensus caspase-3 cleavage site (DxxD)
and is conserved in both human and mouse GSDMD. The
physiological consequence of this cleavage remains to be
investigated. Cleavage of DFNA5 by caspase-3 was very efficient
and complete, and appears to occur only at a single site because
only two fragments (DFNA5-N and DFNA5-C) were generated
when the cleaved products were visualized by Coomassie stain
(Fig. 1b, right panel).
To identify the caspase-3 recognition site in DFNA5 we
searched for a consensus caspase-3 recognition motif (DxxD) in
the linker region between the DFNA5-N and DFNA5-C domains.
We found that both human and mouse DFNA5 proteins contain
putative caspase-3 recognition motifs at residues 267–270
(267DMPD270) (Fig. 1c). To confirm that this site is indeed
cleaved by caspase-3, we substituted Asp270 with Glutamate in
human DFNA5 by site-directed mutagenesis. The resulting D/E
mutant DFNA5 was completely resistant to cleavage by caspase-3
(Fig. 1d). We further subjected the DFNA5-C fragment to Edman
degradation and obtained the N-terminal sequence AAHGI,
which exactly matches the N-terminal sequence of the predicted
DFNA5-C fragment generated by cleavage of human DFNA5
after Asp270.
To show that DFNA5 is a physiological target for caspase-3
after its activation by the Apaf-1 apoptosome, we stimulated S100
cell extracts from 293T cells stably expressing a C-terminal
EGFP-tagged WT or D270E DFNA5 proteins (293T-DFNA5-
EGFP cells or 293T-DFNA5-D270E-EGFP cells, respectively)
with cytochrome c. Notably, activation of endogenous caspase-3
within the Apaf-1 apoptosome by cytochrome c resulted in robust
processing of WT DFNA5 but not the D270E DFNA5 mutant
(Fig. 2a). Caspase-3 activation by cytochrome c in S100
extracts from 293T cells stably expressing GSDMD-EGFP
(293T-GSDMD-EGFP cells) also resulted in processing of
GSDMD at Asp87 (Fig. 2b). No DFNA5 or GSDMD processing
was observed in S100 lysates from the caspase-3-deficient MCF7
breast cancer cell line
26
, indicating that caspase-3 is the primary
protease responsible for processing of DFNA5 and GSDMD
downstream of the apoptosome (Supplementary Fig. 3b, left
panels, and Supplementary Fig. 3c). Consistent with this, DFNA5
processing was restored in MCF-7 cells stably expressing
caspase-3 (Supplementary Fig. 3b, right panels). Similarly,
activation of S100 extracts from HEPG2 cells which unlike
293T cells express detectable endogenous DFNA5 protein
(Fig. 2c), or from immortalized caspase-1/11-double knockout
macrophages, resulted in processing of endogenous DFNA5
and GSDMD (Fig. 2d,e). Combined, these results indicate that
caspase-3 specifically cleaves DFNA5 after Asp270 and that
DFNA5 is a physiological target of caspase-3 downstream of the
Apaf-1 apoptosome.
The processed DFNA5-N fragment has a necrotic activity.To
investigate whether the caspase-3-generated DFNA5-N fragment
has a necrotic activity like the GSDMD-N fragment that is
generated by inflammatory caspases, full-length DFNA5 and
GSDMD and their processed fragments DFNA5-N and
GSDMD-N, respectively, were ectopically expressed in 293T cells.
In contrast to full-length proteins, both DFNA5-N and
GSDMD-N fragments induced extensive cell death with char-
acteristic morphological and biochemical features of necrosis as
evidenced by ballooning of the cell membrane and LDH release,
respectively (Fig. 3a,b). Because of their potent killing activity the
expression levels of DFNA5-N and GSDMD-N fragments were
barely detectable compare to full-length proteins (Fig. 3c).
kDa
70 -
55 -
35 -
27 -
15 -
100 -
DFNA5-N
DFNA5-C
Casp-3
Control
a b
c
DFNA5 FL
+T7-FL
DFNA5
FL
Casp-1
Casp-3
Casp-1
Casp-3
p30
+T7-FL
GSDMD
p13
15 -
35 -
55 -
70 -
Casp-3 Casp-3
WT DFNA5 DFNA5-
D270E
DFNA5 FL
35 -
55 -
DFNA5-N
Caspase-3 cleavage
DFNA5-N DFNA5-C
1 270 496
DFNA5-N DFNA5-C
1 270 512
Human DFNA5
Mouse DFNA5
d
––
kDa
70 -
55 -
35 -
27 -
15 -
100 -
Induced
Un-induced
DFNA5 FL
Figure 1 | Caspase-3 cleaves DFNA5 after Asp270. (a) Immunoblot of purified N-terminal His6-T7-tagged DFNA5 and GSDMD proteins incubated
without (  ) or with caspase-1 (casp-1) or caspase-3 (casp-3) for 45 min at 37 °C. The blot was probed with anti-T7 antibody. (b) Coomassie stained
SDS-polyacrylamide gels of TALON-immobilized proteins from uninduced or IPTG-induced BL-21 pET28b-DFNA5 bacteria (left panel), or IPTG-induced
BL-21 pET28b-DFNA5 bacteria incubated without (control) or with caspase-3 for 45 min (right panel). The caspase-3-generated N- and C-terminal DFNA5
fragments (DFNA5-N and DFNA5-C, respectively) are indicated. (c) Diagrammatic representation of human and mouse DFNA5 proteins showing the
caspase-3 recognition motif at aa 267–270. (d) Immunoblot of purified N-terminal T7-tagged WT DFNA5 and DFNA5-D270E proteins incubated without
(  ) or with increasing amounts of caspase-3 for 45 min. The blot was probed with anti-T7 antibody. Results are representative of at least three
independent experiments.
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14128 ARTICLE
NATURE COMMUNICATIONS | 8:14128 | DOI: 10.1 038/ncomms14128 | www.nature.com/naturecommunications 3