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Zhou et al. Cell Death and Disease (2021) 12:211
https://doi.org/10.1038/s41419-021-03493-2 Cell Death & Disease
ARTICLE Open Access
Hypothermic oxygenated perfusion inhibits
HECTD3-mediated TRAF3 polyubiquitination to
alleviate DCD liver ischemia-reperfusion injury
Wei Zhou
1
, Zibiao Zhong
1
,DanniLin
2
, Zhongzhong Liu
1
, Qiuyan Zhang
1
,HaoyangXia
1
, Sheng Peng
1
,
Anxiong Liu
1
, Zhongshan Lu
1
, Yanfeng Wang
1
,ShaojunYe
1
and Qifa Ye
1,3
Abstract
Ischemia-reperfusion injury (IRI) is an inevitable and serious clinical problem in donations after heart death (DCD) liver
transplantation. Excessive sterile inflammation plays a fateful role in liver IRI. Hypothermic oxygenated perfusion (HOPE), as an
emerging organ preservation technology, has a better preservation effect than cold storage (CS) for reducing liver IRI, in
which regulating inflammation is one of the main mechanisms. HECTD3, a new E3 ubiquitin ligase, and TRAF3 have an
essential role in inflammation. However, little is known about HECTD3 and TRAF3 in HOPE-regulated liver IRI. Here, we aimed
to investigate the effects of HOPE on liver IRI in a DCD rat model and explore the roles of HECTD3 and TRAF3 in its
pathogenesis. We found that HOPE significantly improved liver damage, including hepatocyte and liver sinusoidal
endothelial cell injury, and reduced DCD liver inflammation. Mechanistically, both the DOC and HECT domains of HECTD3
directly interacted with TRAF3, and the catalytic Cys (C832) in the HECT domain promoted the K63-linked polyubiquitination
of TRAF3 at Lys138. Further, the ubiquitinated TRAF3 at Lys138 increased oxidative stress and activated the NF-κB
inflammation pathway to induce liver IRI in BRL-3A cells under hypoxia/reoxygenation conditions. Finally, we confirmed that
the expression of HECTD3 and TRAF3 was obviously increased in human DCD liver transplantation specimens. Overall, these
findings demonstrated that HOPE can protect against DCD liver transplantation-induced-liver IRI by reducing inflammation
via HECTD3-mediated TRAF3 K63-linked polyubiquitination. Therefore, HOPE regulating the HECTD3/TRAF3 pathway is a
novel target for improving IRI in DCD liver transplantation.
Introduction
Owing to the shortage of donor organs, marginal
donor allografts are increasing becoming the main
source of grafts
1
. Particularly, liver allografts from
donations after cardiac death (DCD) may increase the
pool of organs by as much as 20%
2
. Unfortunately, warm
ischemia, cold ischemia, and subsequent reperfusion
phases during DCD liver transplantation can lead to
inevitable ischemia-reperfusion injury (IRI), which
results in delayed graft dysfunction and ultimately
decreases long-term graft survival
3–5
. The ischemic
phase can activate innate immune cells to initiate the
tissue repair process required to restore homeostasis and
provide defense against microbial invasion. However, in
the subsequent reperfusion phase, the excessive activa-
tion of immune responses causes oxidative stress and,
local and systemic inflammation, thereby aggravating
liver damage
6,7
. To alleviate IRI, dynamic preservation
© The Author(s) 2021
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Correspondence: Shaojun Ye (86987100@qq.com) or Qifa Ye (yqf_china@163.
com)
1
Zhongnan Hospital of Wuhan University, Institute of Hepatobiliary Diseases of
Wuhan University, Transplant Center of Wuhan University, Hubei Key
Laboratory of Medical Technology on Transplantation, Engineering Research
Center of Natural Polymer-based Medical Materials in Hubei Province, Wuhan,
China
2
The First Affiliated Hospital, Zhejiang University School of Medicine,
Department of Hepatobiliary and Pancreatic Surgery, Zhejiang Provincial Key
Laboratory of Pancreatic Disease, Innovation Center for the Study of Pancreatic
Diseases, Hangzhou, China
Full list of author information is available at the end of the article
These authors contributed equally: Shaojun Ye, Qifa Ye
Edited by A. Stephanou
Official journal of the Cell Death Differentiation Association
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technology for grafts has been developed. Specifically,
hypothermic oxygenated perfusion (HOPE) has been
demonstrated to decrease oxidative stress and cellular
inflammation in DCD liver transplantation
1,8–10
.How-
ever, little is known about its mechanism of action.
Ubiquitin modification plays an indispensable role in
regulating inflammation and immune responses
11
.
Ubiquitin-protein ligases (E3s) are commonly divided into
two types: HECT and RING
12
.HECTligasescomprise28
members grouped into three subfamilies
12
. The homo-
logous to the E6-associated protein carboxyl terminus
domain containing 3 (HECTD3), a member of the third
subfamily of HECT ligases, is strongly expressed in the
human liver
13
. HECTD3 consists of 861 amino acid resi-
dues, with a DOC domain (219–397) at the N-terminus
and a HECT domain (512–857) at the C-terminus
14
.After
aspecific substrate combines with the DOC domain, it is
usually ubiquitinated and modified via a ubiquitin-HECT
thioester complex
11,14
, thereby exerting its biological
activities. Itch, a HECT E3 ubiquitin ligase, promotes the
degradation of RORγt via K48-linked polyubiquitination to
suppress colonic inflammation
15
. In addition, HECTD3
promotes nondegradative K27-linked and K29-linked
polyubiquitination of Malt1 at K648 and K27-linked poly-
ubiquitination of Stat3 at K180 to activate the pathogenic
Th17 lineage, thereby aggravating the severity of experi-
mental autoimmune encephalomyelitis (EAE) in mice
16
.
These results show that HECTD3 plays a vital role in
coordinating inflammation and immune responses via
ubiquitination, and that it regulates ubiquitination through
different sites depending on substrates. However, whether
HECTD3 regulates inflammation in liver IRI is unclear.
Tumor necrosis factor receptor-associated factor 3
(TRAF3), the other type of E3s, is a representative member
of the TRAF family. As an adaptor molecule, TRAF3
associates the TNF receptor family with numerous signal-
ing pathways related to cell survival and stress responses
17
.
However, owing to the postnatal lethality of global TRAF3-
deficiency, the functions of TRAF3 are identified until the
application of gene conditional knockout
18,19
.Mainly,
TRAF3 is involved in inflammation and immune responses
by regulating NF-κB, MAPK, and type I interferon (IFN-I)
pathways
20,21
. Upon bacterial infection, HECTD3 pro-
motes the polyubiquitination of TRAF3, leading to the
promotion of type I IFN production and inflammatory
response
11
. However, it is unclear whether HETCD3 and
TRAF3 interact directly during bacterial inflammation. In
addition, whether their interaction in sterile inflammation
is unknown. Although a previous study has revealed that
TRAF3 may be involved in the regulation of liver damage
and inflammation by activating the NF-κB pathway during
liver IRI
18
,thespecific action site of TRAF3 and its reg-
ulator are still unclear.
Given the effect of HOPE on inflammation in DCD liver
transplantation and the interaction between HECTD3 and
TRAF3 during bacterial inflammation, we speculated that
HOPE may regulate the inflammatory injury induced by
ischemia-reperfusion (IR) via HECTD3/TRAF3 pathway
in DCD liver transplantation. Herein, in this study, we
sought to investigate that the effect of HOPE on liver IRI
in a DCD rat model and explore the mechanisms of action
of HECTD3 and TRAF3 in its pathogenesis, thereby
seeking a new therapeutic target for improving IRI in
DCD liver transplantation.
Results
HOPE improves liver function and alleviates DCD liver
injury in rats
To verify the effect of HOPE on liver function in a DCD
rat model, we measured alanine aminotransferase (ALT)
and aspartate aminotransferase (AST) activities in perfu-
sate. Their activities were significantly increased in the CS
group compared with the sham group, whereas these
increases were suppressed in the HOPE group (Fig. 1A, B).
Moreover, haematoxylin-eosin (H&E) staining of the liver
tissues and their histological scores confirmed that HOPE
can reduce liver IRI in a DCD rat model (Fig. 1Ea, F).
To explore the effect of HOPE on oxidative stress in liver
IRI, we first tested the activities of malondialdehyde (MDA)
and superoxide dismutase (SOD) in liver tissues. Compared
with the sham group, the CS group showed a distinct
increase in MDA activity, and this increase was suppressed
in the HOPE group (Fig. 1C). Conversely, that of SOD was
higher in the HOPE group than in the CS group (Fig. 1D).
Transmission electron microscope (TEM) results showed
the deep staining of the nuclear chromatin, clear dis-
appearance of the mitochondrial cristae, and large inter-
membrane space between the inner and outer mitochondrial
membranes; these changes were the most prominent in the
CS group (Fig. 1Eb). In addition, the swelling of the smooth
endoplasmic reticulum (SER) and increased rough endo-
plasmic reticulum (rER) were more pronounced in the CS
group than in the HOPE group (Fig. 1Eb), indicating that
HOPE could inhibit oxidative stress in a DCD rat model.
To explore the effect of HOPE on liver sinusoidal
endothelial cells (LSECs), we performed immunohis-
tochemistry (IHC) and immunofluorescence (IF) staining
of CD31 and CD206. The staining results showed that the
expression levels of CD31 and CD206 were increased in
the CS group compared with the sham group, whereas
this increase was inhibited in the HOPE group (Fig. 1Ec,
Ed, G, H), suggesting that HOPE could improve the
LSECs damage-induced by liver IRI.
Collectively, these findings indicate that HOPE could
alleviate DCD liver IRI, including the damage to hepato-
cyte and LSECs.
Zhou et al. Cell Death and Disease (2021) 12:211 Page 2 of 17
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HOPE reduces DCD liver inflammation in rats
To explore the underlying mechanism of the protective
effect of HOPE in DCD liver IRI, we conducted IF staining
of Ly6G, MPO, and CD11b, which are markers of neu-
trophil infiltration. Aggregation of neutrophils was sig-
nificantly higher in the CS group than that in the HOPE
group (Fig. 2A, B). Moreover, compared with the CS
group, HOPE inhibited the expression of interleukin (IL)-
1βand IL-6 (Fig. 2C, D). However, that of IL-10 was
higher in the HOPE group than in the CS group (Fig. 2E).
The messenger RNA (mRNA) levels of tumor necrosis
factor-α(TNF-α) and IL-1βwere higher in the CS group
than in the HOPE group, whereas that of IL-10 showed
the opposite result (Fig. 2F–H). Taken together, these
data suggest that HOPE reduces DCD liver IRI in rats may
by inhibiting inflammation.
HOPE inhibits the expression of HECTD3
Next, we evaluated the expression of HECTD1,
HECTD2, and HECTD3 in liver tissues via western blot.
The expression levels of HECTD1, HECTD2, and
HECTD3 were evidently higher in the CS and HOPE
groups than in the sham group, however, those of
HECTD1 and HECTD2 were not significantly different
Fig. 1 HOPE improves liver function and alleviates DCD liver injury in rats. A, B ALT and AST activities in the perfusate. Data are mean ± SD, **P
< 0.01 by one-way ANOVA followed by Tukey’s test. n =6 per group. C, D MDA and SOD activities in rat liver tissues. Data are mean ± SD, **P< 0.01
by one-way ANOVA followed by Tukey’s test. n =6 per group. E (a) Histological damage in liver of rats. H&E staining was performed on paraffin-
embedded section of rat liver tissues. Scale bar =200 μm (left panels) and 50 μm (right panels). E (b) Changes in the microstructure of hepatocytes.
The changes of hepatocellular microstructure were observed using TEM. N, nucleus; M, mitochondria; r, rough endoplasmic reticulum; S, smooth
endoplasmic reticulum. The black arrows indicate the lacuna between the inner and outer membranes of mitochondria. Scale bar =2μm (left
panels) and 500 nm (right panels). E (c, d) Activation of LSECs. IHC staining (c) and IF staining (d) of CD31 and CD206 were performed on paraffin-
embedded section of rat liver tissues. Scale bar =100 μm. FHistological scores were analyzed based on Suzuki’s criteria. Data are mean ± SD,
**P< 0.01 by one-way ANOVA followed by Tukey’s test. n=6 per group. G, H IHC and IF stained fluorescence of CD31 and CD206 were quantified
using Image-pro plus 6.0. Data are mean ± SD, *P< 0.05 and **P< 0.01 by two-way ANOVA followed by Tukey’s test. n=6 per group.
Zhou et al. Cell Death and Disease (2021) 12:211 Page 3 of 17
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between the CS and HOPE groups, except for HECTD3
(Fig. 3A, B). Similarly, their mRNA levels showed this
trend (Fig. 3C–E). IHC results further verified that the
expression level of HECTD3 in the HOPE group was
significantly lower than that in the CS group (Fig. 3F, G).
Overall, these suggest that HOPE can inhibit the expres-
sion of HECTD3, but not HECTD1 and HECTD2, com-
pared with CS in a DCD rat model.
HOPE reduces DCD liver injury and inflammation by
inhibiting HECTD3 in rats
To further explore whether HOPE reduces liver IRI and
inflammation by targeting HECTD3, we reconstructed
and in vivo transfected adeno-associated virus 8 (AAV8)
to regulate HECTD3, including the HECTD3-
overexpression (ov-HECTD3), AAV8 to inhibit its
expression (sh-HECTD3) and the corresponding AAV8
empty virus (AAV8 NC). We found that the fluorescence
intensity of GFP was the highest on day 12 after trans-
fection (Fig. S1A, B). Moreover, the transfection of AAV8
NC had no significant effect on ATL and AST activities
(Fig. S1C, D). Furthermore, compared with the sham and
AAV8 NC pre-treatment groups, the ov-HECTD3 AAV8
transfected group had significantly increased HECTD3
expression, whereas the sh-HECTD3 AAV8 transfection
group inhibited HECTD3 expression (Fig. S1E). There-
fore, in subsequent experiments, we determined the
transfection time to be 12 days.
Fig. 2 HOPE reduces DCD liver inflammation in rats. A IF staining of Ly6G, MPO and CD11b were performed on paraffin-embedded section of rat
liver tissues. Scale bar =100 μm. BIF stained fluorescence of Ly6G, MPO and CD11b were quantified using Image-pro plus 6.0. Data are mean ± SD,
*P< 0.05 and **P< 0.01 by two-way ANOVA followed by Tukey’s test. n=3 per group. C–ELiver tissues expression levels of IL-1β, IL-6, and IL-10 were
analyzed with the relative ELISA kits. Data are mean ± SD, **P< 0.01 by one-way ANOVA followed by Tukey’s test. n=6 per group. F–HmRNA
expression level in liver of TNF-α, IL-1β, and IL-10 were tested by real-time PCR. Data are mean ± SD, n.s., not significant; *P< 0.05 and **P< 0.01 by
one-way ANOVA followed by Tukey’s test. n=3 per group.
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To explore the role of HECTD3 in HOPE in improving
DCD liver IRI, we examined the ALT and AST activities
in the perfusate after transfection. ALT and AST activities
significantly increased after pre-treatment with ov-
HECTD3 AAV8, whereas that with sh-HECTD3 AAV8
had the opposite effect (Fig. 4A, B). However, compared
with the CS group, the HOPE group pretreated with ov-
HECTD3 AAV8 reversed the effects of HOPE (Fig. 4A, B).
Consistently, compared with the HOPE group, ALT and
AST activities were not significantly different in the CS
group pretreated with sh-HECTD3 AAV8 (Fig. 4A, B).
Meanwhile, we confirmed that ov-HECTD3 and sh-
HECTD3 AAV8 had no effect on ALT and AST activ-
ities (Fig. S1F, G). In line with this, H&E staining of the
liver tissues and the histological scores confirmed that
pre-treatment with ov-HECTD3 AAV8 aggravated liver
damage, whereas that with sh-HECTD3 AAV8 visibly
alleviated liver damage (Fig. 4C, D).
Subsequently, we detected the levels of IL-1β, IL-6, and
IL-10 in liver tissues after transfection. Enzyme-linked
immunosorbent assays (ELISA) results suggested that
pre-treatment with ov-HECTD3 AAV8 promoted the
expression of IL-1βand IL-6 but inhibited that of IL-10;
however, pre-treatment with sh-HECTD3 AAV8 had the
opposite effects (Fig. 4E–G). We further verified that ov-
HECTD3 and sh-HECTD3 AAV8 had no effect on the
expression of IL-1β, IL-6, and IL-10 (Fig. S1H–J).
Therefore, our data showed that HOPE reduced liver IRI
and inflammation by targeting HECTD3 in a DCD
rat model.
Fig. 3 HOPE inhibits the expression of HECTD3 in a DCD rat model. A, B HECTD1, HECTD2, and HECTD3 proteins expression in liver were
evaluated using western blot (A) and quantified using a Gel-Pro Analyzer (B). Data are mean ± SD, n.s., not significant; **P< 0.01 by two-way ANOVA
followed by Tukey’s test. n=3 per group. C–EmRNA expression level in liver of HECTD1, HECTD2 and HECTD3 were tested by real-time PCR. Data are
mean ± SD, n.s., not significant; *P< 0.05 and **P< 0.01 by one-way ANOVA followed by Tukey’s test. n=3 per group. FIHC staining of HECTD3 were
performed on paraffin-embedded section of rat liver tissues. Scale bar =100 μm (upper panels) and 50 μm (down panels). GIHC stained fluorescence
of HECTD3 was quantified using Image-pro plus 6.0. Data are mean ± SD, *P< 0.05 and **P< 0.01 by one-way ANOVA followed by Tukey’s test. n=6
per group.
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HOPE regulates TRAF3 polyubiquitination by targeting
HECTD3
To explore the underlying mechanism of HECTD3
reduction in liver IRI and inflammation, we next analyzed
the expression and activation of TRAF3. Western blot
analysis indicated that the expression of TRAF3 was dis-
tinctly increased in the CS group, and this effect was
suppressed in the HOPE group (Fig. 5A). Furthermore, we
found that TRAF3 polyubiquitination was remarkably
increased in the CS group; however, this effect was
inhibited in the HOPE group (Fig. 5B). These suggest that
HOPE suppressed the polyubiquitination of TRAF3 in a
DCD rat model.
To confirm whether HECTD3 and TRAF3 interacts in the
DCD rat model, we performed co-immunoprecipitation
(Co-IP) analysis. Compared with the CS group, HOPE partly
inhibited the interaction between HECTD3 and TRAF3 (Fig.
5C). To further verify this interaction, we established a
hypoxia/reoxygenation (H/R) model of BRL-3A cells and a
cold storage/reoxygenation (CS/R) model of LSECs to
simulate in vivo warm IR and cold IR, respectively. Com-
pared with the control group, H/R and CS/R visibly pro-
moted the co-localization of HECTD3 and TRAF3 in BRL-
3A cells and LSECs, respectively (Fig. 5D,E).Therefore,our
in vivo and in vitro experiments confirmed the inevitable
interaction between HECTD3 and TRAF3.
Fig. 4 HOPE reduces DCD liver IRI and inflammation by inhibiting HECTD3 in rats. Rats were infected with ov-HECTD3 or sh-HECTD3 AAV8. The
rats in the sham group were infected with or without AAV8 NC served as the negative control. After the specimen collection, the following analysises
were performed. A,BALT and AST activities in perfusate. Data are mean ± SD, n.s., not significant; *P< 0.05 and **P< 0.01 by one-way ANOVA
followed by Tukey’s test. n=5 per group. CHistological damage in liver of rats. H&E staining was performed on paraffin-embedded section of rat
liver tissues. Scale bar =100 μm. DHistological scores were analyzed according to Suzuki’s criteria. Data are mean ± SD, n.s., not significant; **P< 0.01
by one-way ANOVA followed by Tukey’s test. n=5 per group. E–GLiver tissues expression levels of IL-1β, IL-6, and IL-10 were analyzed with the
relative ELISA kits. Data are mean ± SD, n.s., not significant; **P< 0.01 by one-way ANOVA followed by Tukey’s test. n=5 per group.
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To further explore whether HECTD3 regulates the
polyubiquitination of TRAF3, we conducted the ubiqui-
tination analysis after rats were transfected with
ov-HECTD3 and sh-HECTD3 AAV8. We found that pre-
treatment with ov-HECTD3 AAV8 promoted the TRAF3
polyubiquitination, whereas pre-treatment with sh-
HECTD3 AAV8 visibly inhibited this effect (Fig. 5F).
Thus, these data suggest that HOPE inhibits the poly-
ubiquitination of TRAF3 by targeting HECTD3.
Interaction between HECTD3 and TRAF3
To clarify the interaction between HECTD3 and TRAF3
in liver IRI, we first showed the schematic diagram of the
domain architecture of HECTD3 (Fig. 6A). Subsequently,
Fig. 5 HOPE inhibits the ubiquitination of TRAF3 by targeting HECTD3. A TRAF3 protein expression in liver was evaluated using western blot
and quantified using a Gel-Pro Analyzer. Data are mean ± SD, **P< 0.01 by one-way ANOVA followed by Tukey’s test. n=3 per group. B,C
Ubiquitination analysis of TRAF3 and Co-IP analysis of the interaction between HECTD3 and TRAF3. Liver tissues protein extracts were IP with primary
antibody for TRAF3 (B) or HECTD3 (C). The immunoprecipitates were blotted with the relative antibodies. D,ECo-localization of HECTD3 and TRAF3
in hepatocyte and LSECs. IF staining of HECTD3 and TRAF3 were examined in BRL-3A cells with or without H/R and examined in LSECs with or
without CS/R. DAPI was used to counter stain nuclei. The stained fluorescence was observed using a fluorescence microscope and quantified using
Image-pro plus 6.0. Scale bar =50 μm. Data are mean ± SD, **P< 0.01 by Student’s two-tailed unpaired t-test. n=10 per group. FUbiquitination
analysis for the regulation of endogenous TRAF3 by HECTD3. Partial groups of rats were infected with ov-HECTD3 or sh-HECTD3 AAV8. Liver tissues
protein extracts were immunoprecipitated with primary antibody for TRAF3. The immunoprecipitates were blotted with the relative antibodies.
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Fig. 6 Interaction between HECTD3 and TRAF3. A Schematic diagram of the domain architecture of HECTD3. The DOC domain is located at the
amino acid (aa) sequence 219 to 397, whereas the HECT domain is located at the amino acid (aa) sequence 512 to 857. BCoomassie-stained SDS-
PAGE of purified TRAF3 and recombinant HECTD3 constructs: GST-tagged TRAF3 and His-tagged full-length HECTD3, His-tagged isolated DOC and
HECT domains. C–EGST pull-down assay with purified GST-TRAF3 and recombinant His-tagged full-length HECTD3, His-tagged isolated DOC and
HECT domains. Purified GST-tagged TRAF3 was incubated with His-tagged full-length HECTD3 (C), the isolated DOC (D) and HECT domains (E). The
interaction between TRAF3 and HECTD3, the DOC and HECT domains were visualized using immunoblots. F–HUbiquitination analysis of TRAF3 and
the interaction sites between HECTD3, Ub and TRAF3. BRL-3A cells transfected with mutants HECTD3 (C823A), HA-tagged Ub (K63R) and FLAG-
tagged TRAF3 (K138R) plasmids or the corresponding WT and vector plasmids. Cellular protein extracts in BRL-3A cells with or without H/R were
immunoprecipitated with primary antibody for FLAG. The immunoprecipitates were blotted with the relative antibodies.
Zhou et al. Cell Death and Disease (2021) 12:211 Page 8 of 17
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glutathione S-transferase (GST) pull-down assays
demonstrated that the GST-TRAF3 protein, but not GST,
was pulled down by exogenous His-HECTD3, His-DOC,
His-HECT (Fig. 6B–E), indicating that both full-length
HECTD3 and isolated DOC and HECT domains directly
interacted with TRAF3.
To pinpoint the binding site between HECTD3 and
TRAF3 and identify the type of TRAF3 polyubiquitination
regulated by HECTD3 in H/R, we conducted the following
experiments. First, to verify the catalytic cysteine (C832) in
the HECT domain essential for TRAF3 polyubiquitination,
we constructed a HECTD3 mutant HECTD3 (C823A).
Subsequent ubiquitination analysis showed that the poly-
ubiquitination level of TRAF3 in the presence of HECTD3
(C823A) was lower than that of HECTD3 in the control
and H/R groups, and that the overall level of in the H/R
group was higher than that of in the control group (Fig. 6F).
Second, to identify the type of TRAF3 polyubiquitination
regulated by HECTD3 in H/R, we constructed a HA-
ubiquitin (Ub) mutant HA-Ub (K63R). Compared with the
group transfected with HA-Ub, that with HA-Ub (K63R)
inhibited the polyubiquitination of TRAF3, and the overall
level of the H/R group was higher than that of the control
group (Fig. 6G). Finally, to identify the lysine (Lys)138
residue is essential for HECTD3-mediated K63-linked
polyubiquitination of TRAF3, we constructed a TRAF3
mutant TRAF3 (K138R). Evidently, TRAF3 poly-
ubiquitination was diminished in K138R in both the con-
trol and H/R groups (Fig. 6H). Overall, these data
suggested that the C832 of the HECT domain promotes
the K63-linked polyubiquitination of TRAF3 at Lys138 in
BRL-3A cells under H/R conditions.
TRAF3 Lys138 is essential for regulating H/R-induced injury
in BRL-3A cells
To explore the role of TRAF3 Lys138 in H/R, we
transfected BRL-3A cells with TRAF3 (WT) and TRAF3
(K138R) plasmids. Through the assays of ALT, AST and
lactate dehydrogenase (LDH) activities in the culture
medium, we found that the ALT, AST, and LDH activities
in the presence of TRAF3 (K138R) were lower than those
of TRAF3 (WT) in the H/R groups, whereas there was no
significant difference in the control groups (Fig. 7A–C).
These findings indicate that TRAF3 Lys138 is essential for
regulating the liver function injury induced by H/R in
BRL-3A cells. Moreover, cell counting kit-8 (CCK-8)
analysis showed that the cell survival rate significantly
increased in the presence of TRAF3 (K138R) than that of
TRAF3 (WT) in the H/R groups (Fig. 7D), indicating that
TRAF3 Lys138 is crucial for cell survival in the H/R of
BRL-3A cells.
To elucidate the mechanism by which the TRAF3
Lys138 mutant attenuated cell death in BRL-3A cells
under H/R conditions, we evaluated the accumulation of
reactive oxygen species (ROS) in BRL-3A cells. There was
a significantly increase in ROS accumulation in the group
transfected with TRAF3 (WT) than in that with TRAF3
(K138R) under H/R conditions (Fig. 7E), suggesting that
TRAF3 Lys138 mutants prominently inhibited oxidative
stress in H/R. In addition, BRL-3A cells in H/R trans-
fected with TRAF3 (K138R) inhibited the phosphorylation
of IKKβ,IκBα, and p65 at sites Tyr199, Ser32/Ser36, and
Ser536, respectively (Fig. 7F–J), which are vital proteins in
the NF-κB signaling pathway. Thus, the TRAF3 Lys138
mutants inhibited the activation of the NF-κB signaling
pathway in BRL-3A cells subjected to H/R. In summary,
the TRAF3 Lys138 mutant is essential for suppressing
oxidative stress and inflammation induced by H/R in
BRL-3A cells.
Human liver IR promotes the expression of HECTD3 and
TRAF3
To verify the changes in HECTD3 and TRAF3 levels in
human livers suffering from IR, we collected five pairs of
DCD liver transplantation specimens. The donor liver
underwent warm and cold ischemia for almost a period of
time and a one-hour reperfusion (Table 1). Through H&E
staining of tissue microarray (TMA) and histological
scores, we found that IR caused varying degrees of
pathological damage to human liver tissues (Fig. 8A, B).
Moreover, the expression of CD31 visibly increased in the
IR group compared with the control group (Fig. 8C, D),
suggesting that the LSECs in human liver tissues were
damaged by IR. Further, western blot results showed that
the expression levels of HECTD3 and TRAF3 in the IR
group were significantly higher than those in the control
group (Fig. 8E, F), suggesting that human liver IR during
DCD liver transplantation promotes the expression of
HECTD3 and TRAF3.
Discussion
IRI during DCD liver transplantation can lead to up to
10% of early transplant failures and increase graft rejec-
tions
22
. We and other researchers have previously
demonstrated that HOPE has a significant improved liver
IRI after transplantation by regulating inflammation
1,8,10
;
however, the underlying protective mechanism is unclear.
Our results of this study demonstrated that HOPE redu-
ces inflammation by regulating the HECTD3/
TRAF3 signaling pathway to improve DCD liver IRI.
Studies on rat models of DCD liver transplantation
showed that warm ischemia can cause significant damage
to hepatocytes, especially mitochondria, and this damage
is aggravated with prolonged ischemia
23
. In this study, we
found that these injuries were alleviated after HOPE
treatment, indicating that HOPE significantly improved
hepatocyte injury in a DCD rat model. In contrast, the
damage caused by cold ischemia mainly targets
Zhou et al. Cell Death and Disease (2021) 12:211 Page 9 of 17
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Table 1 Donor and recipient information for liver transplantation.
Donor Recipient
Number Age
(years)
Gender Total ischemic
time (h)
Warm ischemia
time (min)
Cold ischemia
time (h)
Age
(years)
Gender Reperfusion
time (h)
1 57 Male 7.4 14 7.2 27 Male 1
2 63 Male 8.2 12 8.0 48 Male 1
3 71 Male 12.0 12 11.8 40 Male 1
4 16 Male 8.0 15 7.7 49 Male 1
5 67 Male 9.7 16 9.4 50 Male 1
Fig. 7 TRAF3 Lys138 mutants reduce the injury of BRL-3A cells with H/R. BRL-3A cells transfected with TRAF3 Lys138 mutants (K138R) plasmids
or the corresponding WT and vector plasmids as indicated, and the cells were cultured under normal or H/R conditions. The following analysises
were performed. A–CALT, AST, and LDH activities in the culture medium. Pretreated BRL-3A cells were incubated under normal or H/R conditions.
Data are mean ± SD, n.s., not significant; **P< 0.01 by two-way ANOVA followed by Tukey’s test. n=3 per group. DCCK-8 assay was used to examine
cell survival. Data are mean ± SD, n.s., not significant; **P< 0.01 by two-way ANOVA followed by Tukey’s test. n=6 per group. EROS in BRL-3A cells.
ROS staining was performed to evaluate the level of ROS in BRL-3A cells, and the stained fluorescence was observed using a fluorescence
microscope. The cellular morphology and structure of BRL-3A cells by bright image investigation. F–JProteins levels of p-IKKβ, IKKβ, p-IκBα,IκBα,p-
p65, p65 in BRL-3A cells were evaluated using western blot (F) and quantified using a Gel-Pro Analyzer (G–J). Data are mean ± SD, n.s., not significant;
**P< 0.01 by two-way ANOVA followed by Tukey’s test. n=3 per group.
Zhou et al. Cell Death and Disease (2021) 12:211 Page 10 of 17
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LSECs
24,25
. Studies have shown that CS can cause mor-
phological changes in LSECs, including the retraction and
detachment of cell bodies. Subsequent reperfusion can
aggravate this damage and result in the almost complete
deprivation of the LSECs lining
25
. Similarly, this study
confirmed that CS can significantly increase the expres-
sion of CD31 and CD206, important markers of LSECs
damage
26–28
. Surprisingly, the damage was visibly
Fig. 8 Human liver IRI promotes the expression of HECTD3 and TRAF3. A, B Histological damage in human liver. Overview of a TMA section
stained with H&E. Histological change was observed using a microscope (A). The right panel is a representative enlarged image. Scale bar =100 μm.
Histological scores were analyzed depending on Suzuki’s criteria (B). Data are mean ± SD, **P< 0.01 by Student’s two-tailed unpaired t-test. n=20
per group. C,DActivation of LSECs in human liver. Overview of a TMA section stained with IHC. The right panel is a representative enlarged image.
Scale bar =100 μm. IHC stained fluorescence of CD31 was quantified using Image-pro plus 6.0. Data are mean ±SD, **P< 0.01 by Student’s two-
tailed unpaired t-test. n=20 per group. E,FHECTD3 and TRAF3 proteins expression in human liver were evaluated using western blot (E) and
quantified using a Gel-Pro Analyzer (F). Data are mean ± SD, n.s., not significant; **P< 0.01 by two-way ANOVA followed by Tukey’s test. n=5 per
group. GMechanism scheme. Warm ischemia, cold ischemia, and subsequent reperfusion phases in a DCD rat model cause the DOC and HECT
domains of HECTD3 directly binding to TRAF3. Followed by the C823 of the HECT domains promotes the K63-linked polyubiquitination of TRAF3 at
Lys138 to trigger the activation of NF-κB signaling pathway, leading to the release of inflammatory factors to result in liver IRI. However, HOPE
effectively improved the entire pathological process.
Zhou et al. Cell Death and Disease (2021) 12:211 Page 11 of 17
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suppressed after HOPE treatment, suggesting that HOPE
can also improve LSECs damage in a DCD rat model.
In previous research, we have verified that HOPE can
reduce liver IRI by inhibiting oxidative stress and
inflammation
8,10
. In addition, a study on fatty liver grafts
showed that HOPE inhibits nuclear damage-associated
molecular patterns (DAMPs) trigger, and the activation of
toll-like receptors and stellate cells, thereby decreasing
cellular inflammation
1
. However, the underlying
mechanism of HOPE in improving liver IRI has not been
thoroughly explored in this study. In the present study, to
better simulate the clinical liver transplantation setting,
we extended the duration of CS and immediately per-
formed HOPE treatment in rat livers. Surprisingly, HOPE
treatment consistently reduced the liver inflammation and
injury induced by long-term CS and the subsequent
reperfusion, thereby confirming that HOPE can improve
DCD liver IRI in rats by targeting inflammation. Fur-
thermore, we found that HECTD3 and TRAF3, two dif-
ferent types of E3s, are indispensable in the regulation of
HOPE for DCD liver IRI in rats.
Presently, little is known about the function of HECTD3
(ref.
29
). According to current research, HECTD3 plays an
important role in regulating tumors, as well as inflam-
mation and immune-related diseases, by interacting with
other proteins
13,16,29–31
. In a study on EAE, HECTD3
promoted the nondegradative polyubiquitination of Stat3
and Malt1 to increase the upregulation of RORγt and the
activation of NF-κB, thus aggravating neuroinflamma-
tion
16
. This indicates that HECTD3 plays a proin-
flammatory role in EAE. Similarly, here, we found that the
overexpression of HECTD3 promoted DCD liver injury
and inflammation in rats, whereas its inhibition had the
opposite effect. Interestingly, the expression of HECTD3,
but not of HECTD1 and HECTD2, was significantly dif-
ferent between the CS and HOPE groups. Structurally, both
HECTD1 and HECTD2 do not contain a unique DOC
domain, which is responsible for binding to the substrate to
play more biological functions
12,14
. Functionally, HECTD1
is responsible for neural tube closure during embryonic
development
32
, whereas HECTD2 may be mainly related to
HPV-induced cervical cancers and Angelman syndrome
33
.
Therefore, HOPE targets HECTD3, but not HECTD1 and
HECTD2, to regulate inflammation, thereby improving the
liver IRI-induced by DCD in rats.
Significant progress has been made in studying the role of
E3s in disease progression during bacterial and viral infec-
tions. Once infected with DNA or RNA viruses and various
bacteria, some RING-type E3s play positive or negative roles
in the pathological processes by regulating different types of
polyubiquitination
11,34,35
. However, the role of HECT-type
E3s remains unclear. Recent studies have shown that
HECTD3 promotes the polyubiquitination of TRAF3 to
modulate type I interferon induction upon infection with
intracellular bacteria
11
. However, the study did not examine
whether HECTD3 directly interacts with TRAF3. Moreover,
the interaction between HECTD3 and TRAF3 remains
undocumented in sterile inflammation, which is a major
clinical concern during liver IRI, can be triggered by various
danger molecules, including DAMPs, pattern recognition
receptors (PRRs), and high mobility group box 1 (HMGB1)
(ref.
7,36
). Here, we found that the HECT domain, which is
mainly responsible for the catalytic function of HECTD3,
can also directly bind to TRAF3, in addition to the DOC
domain. However, another study showed that only the DOC
domain directly combines with CRAF and HSP90 (ref.
30
).
Hence, the function of the HECT domain of HECTD3 may
vary depending on the specific substrate. Further, we
verified that the C823 of the HECT domain promoted
the K63-linked polyubiquitination of TRAF3 to regulate
inflammation in BRL-3A cells under H/R conditions.
Therefore, the interaction between HECTD3 and TRAF3
plays an important role in regulating inflammation in both
bacterial and sterile conditions.
As a representative member of RING-type E3s, TRAF3
plays an indispensable role in regulating immune and
inflammation-related diseases. Ubiquitin modification is
the main form of TRAF3 activation. The polyubiquitin
chains link to lysine residues mainly including K6, K11,
K27, K29, K33, K48, and K63 (ref.
37
), depending on the
specific context, thereby playing different roles. For
instance, K48-linked ubiquitination is responsible for
substrate degradation, whereas K63-linked ubiquitination
is responsible for downstream signaling activation and
protein trafficking
38
. Specifically, cIAPs, an E3 ubiquitin
ligase, promotes K48-linked polyubiquitination of TRAF3
for proteasomal degradation, which is a novel mechanism
for paricalcitol, a vitamin D receptor agonist, to inhibit
inflammation in renal diseases
39
. Moreover, Mint3, a
unique member of the Mint protein family, promotes the
K63-linked polyubiquitination of TRAF3 to enhance IRF3
activation and IFN-βproduction induced by TLR3/4 and
RIG-I (ref.
40
). Overtly, different regulators of TRAF3 have
different effects through various lysine residues linking
polyubiquitination of TRAF3 under different conditions.
In this study, we showed that increased HECTD3 pro-
moted the K63-linked polyubiquitination of TRAF3 at
Lys138 in BRL-3A cells subjected to H/R. Moreover, this
increased polyubiquitination aggravated liver function
and reduced the survival of BRL-3A cells under H/R
conditions by regulating oxidative stress and the NF-κB
signaling pathway. These data suggest that targeting
HECTD3 regulated K63-linked polyubiquitination of
TRAF3 at Lys138 may be an important mechanism for
regulating the injury of BRL-3A cells during H/R. Finally,
the increased expression of HECTD3 and TRAF3 was
confirmed in human liver specimens subjected to IRI in
DCD liver transplantation. Unfortunately, we could not
Zhou et al. Cell Death and Disease (2021) 12:211 Page 12 of 17
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perform HOPE treatment on theses human specimens
because the HOPE system applied in the clinical setting is
still under investigation.
In conclusion, we showed that the HECTD3/
TRAF3 signaling pathway played an essential role in
HOPE in improving liver IRI by inhibiting inflammation
in a DCD rat model. Particularly, upon DCD liver IR
insult, the DOC and HECT domains of HECTD3 directly
bind to TRAF3, and the C832 of the HECT domain
promotes the K63-linked polyubiquitination of TRAF3 at
Lys138 to trigger oxidative stress and inflammatory
response, thereby leading to serious liver IRI. However,
HOPE effectively improved the entire pathological pro-
cess. Therefore, HOPE targeting the HECTD3/
TRAF3 signaling pathway represents a promising ther-
apeutic target for liver IRI in DCD liver transplantation.
Materials and methods
Establishment of DCD rat model
Adult male Sprague-Dawley (SD) rats (8- to 10-week-
old; weight: 250–300 g) were obtained from Wuhan Wan
Qian Jia Xing Bio-Technology Co., Ltd. (Hubei, China).
The rats were provided with suitable food and water and
housed in an environment with controlled humidity,
temperature, and light (12:12 light/dark cycle).
The DCD rat model was generated as previously descri-
bed
5,8
.Briefly, the rats were randomly divided into different
groups and the investigator was not blinded to group allo-
cation during the experiment. The rats were administered
with anesthesia via intraperitoneal injection of pentobarbital
sodium salt (30 mg/kg body mass) before midline lapar-
otomy. Subsequently, cardiac death was caused by hypoxia
after diaphragm incision without prior heparinization or
portal clamping
41
. The in situ warm ischemia phase starts
from cardiac arrest. During this phase, the liver temperature
wasmaintainedat29±1.45°C.After30min,toflush out
the blood in the livers, 50 mL of 4 °C histidine-tryptophan-
ketoglutarate (HTK) solution (Dr. Franz Koehler Chemie
GmbH, Bensheim, Germany) was used to perfuse the livers
in situ through the abdominal aorta. After the perfusion, the
portal vein and superior hepatic caval vein were cannulated,
and the infrahepatic vena caval and right adrenal vein were
ligated. Finally, the liver was resected.
Subsequently, the isolated livers were subjected to 23 h
of CS, and some samples were treated using HOPE for
1 h, whereas the others were not. Then, all livers were
subjected to 1 h of normothermic perfusion (NMP). In the
sham group, the isolated healthy livers were only sub-
jected to 1 h of NMP without warm ischemia and CS. The
HOPE and isolated perfusion rat liver model (IPRL) sys-
tems were described in detail in a previous study
8
.
The study protocol was approved by the Ethical Com-
mittee of Wuhan University, and all animal experiments
were carried out in accordance to the Experimental
Animal Management Ordinance (National Science and
Technology Committee of China) and the Guide for the
Care and Use of Laboratory Animals (National Institutes
of Health, Bethesda, MD, USA).
AAV8 construction and in vivo transfection
Recombinant AAV8 for HECTD3-overexpression (ov-
HECTD3), AAV8 to inhibit its expression (sh-HECTD3),
and the homologous AAV8 empty virus (AAV8 NC) were
purchased from Genechem Biotech Inc. (Shanghai, China).
AAV8 was administered via tail-vein injection at a dose of
1.0 × 10
11
viral genome (v.g.) per rat (1 mL total volume).
Cell culture and H/R or CS/R model
BRL-3A cells and LSECs (BNBIO., Beijing, China) were
cultured in a humidified incubator (Thermo, Marietta,
GA, USA) maintained at 37 °C and 5% CO
2
in high-
glucose Dulbecco’s Modified Eagle Medium (DMEM)
supplemented with 10% heat-inactivated fetal bovine
serum, 1% 100 U/mL penicillin G, and 100 μg/mL strep-
tomycin. To generate the in vitro H/R model, BRL-3A
cells were incubated for 12 h in a microaerophilic system
(Heal Force, Shanghai, China) containing 5% CO
2
,1%O
2
,
and 94% N
2
gas. Then, the cells were cultured for 6 h
under normoxic conditions to allow reoxygenation.
Meanwhile, to generate the in vitro CS/R model, LSECs
were cultured for 24 h at 4 °C in cold Celsior or University
of Wisconsin (UWS) solutions instead of the normal
medium. Subsequently, they were washed twice with cold
phosphate-buffered saline (PBS) and cultured for 2 h
under normoxic conditions to mimick reperfusion
24
.
Plasmid construction and in vitro transfection
The TRAF3 (WT), HECTD3 (WT) plasmid, FLAG-
tagged TRAF3 plasmid, HA-tagged Ub plasmid, HECTD3
(C823A) plasmid (whose cysteine residue at position 823
was replaced by alanine), HA-tagged Ub (K63R) plasmid
(whose lysine residue at position 63 was replaced by
arginine), FLAG-tagged TRAF3 (K138R) plasmid (whose
lysine residue at position 138 was replaced by arginine),
and the empty vector plasmid were synthesized by Gen-
echem Biotech Inc. (Shanghai, China). The BRL-3A cells
were transfected with the relevant plasmid or the empty
vector plasmid using Lipofectamine 3000 according to the
manufacturer’s instructions.
Biochemical analysis
Blood was collected from the postcava and centrifuged
at 3500 rpm for 10 min. The perfusate and liver tissues
were collected after normothermic reperfusion, and the
serum, perfusate, and liver tissues were stored at −80 °C.
The cell culture medium was collected after normal cul-
ture or modeling. ALT and AST levels in the rat serum
and perfusate were measured using automatic analysis in
Zhou et al. Cell Death and Disease (2021) 12:211 Page 13 of 17
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the Zhongnan Hospital of Wuhan University. MDA and
SOD activities in the rat liver were measured using
commercial kits (Jiancheng, Nanjing, China) according to
the manufacturer’s protocol. The activities of the AST,
ALT, and LDH in the cell culture medium were measured
using an automatic biochemical analyzer (Rayto, Shenz-
hen, China).
H&E staining
Liver tissues were harvested, fixed in 4% paraformaldehyde
and embedded in paraffin as previously described
42
.All
paraffin sections were stained with H&E for histological
observation and the graded blindly according to Suzuki’s
criteria
43
. Histological changes were graded from 0 to 4
depended on the severity of cellular vacuolization, hepatic
sinusoid congestion, and hepatocyte necrosis.
TEM assay
Fresh liver tissue fragments (1–2mm
3
) were immediately
fixed in 2.5% glutaraldehyde overnight at 4 °C, washed with
0.1 M PB three times for 30 min each, fixed with 2%
osmium tetroxide in 0.1M PB for 2 h, and dehydrated in
graded ethanol solutions (30, 50, 70, 80, 85, 90, 95, and
100%) for 15 min at each concentration. Then, they were
infiltrated with propylene oxide (PO) twice for 20 min each
and placed into a 70:30 mixture of PO and resin for 1 h. PO
was volatilized by keeping the lid of the tube open over-
night. The samples were transferred to fresh 100% resin,
polymerize at 60 °C for 48 h, cut into ultra-thin sections
(70–90 mm), stained with 2% uranyl acetate at room
temperature for 15 min, washed with distilled water, and
further stained with lead stain solution at room tempera-
ture for 3 min. The grids were observed using a transmis-
sion electron microscope (Tecnai G
2
20 TWIN; FEI,
Hillsboro, USA) at an acceleration voltage of 200 kV.
IHC and IF staining
For IHC, the liver tissues were fixed in 4% paraf-
ormaldehyde, embedded in paraffin, and sectioned (4-μm
thickness). They were then dewaxed, hydrated for 30 min at
room temperature, and incubated overnight at 4 °C with the
following primary antibodies: anti-CD31 (1:1000, Pro-
teintech, Wuhan, China); anti-CD206 (1:10000, Proteintech,
Wuhan, China), anti-HECTD3 (1:400, BIOS, Beijing, China).
Subsequently, the slides were incubated with a horseradish
peroxidase-labeled secondary antibody, and the immunor-
eactivity was visualized with 3,3-diaminobenzidine tetra-
hydrochloride (DAB). Tissue sections were counterstained
with hematoxylin and visualized using Leica Microsystems
at 200 × and 400 × magnification. Image-pro plus 6.0 was
used for image analysis (Media CybernetiHOPE, Inc.,
Rockville, MD, USA).
IF was performed as previously reported
44
.Specifically,
the sections or cell slides were incubated with anti-CD31,
anti-CD206, anti-TRAF3 (1:100, Proteintech, Wuhan,
China), anti-Ly6G (1:100, Novus, Littleton, USA), anti-MPO
(1:200, Servicebio, Wuhan, China), anti-CD11b (1:500, Ser-
vicebio, Wuhan, China) and anti-HECTD3 (1:100, BIOS,
Beijing, China) antibodies. After washing, the slides were
incubated with Alexa Fluor 594-conjugated secondary
antibodies or Alexa Fluor 488-conjugated secondary anti-
bodies (1:200, Proteintech, Wuhan, China).
Enzyme-linked immunosorbent assays (ELISA)
ELISA commercial kits (MultiSciences Biotech, Hang-
zhou, China) were used to measure the IL-1β, IL-6, and
IL-10 concentrations in rat liver tissues according to the
manufacturer’s instructions. The optical density of each
well was determined using a microplate reader (Molecular
Devices, CA, USA). The detailed description of the spe-
cific protocol has been reported previously
45
.
Quantitative real-time PCR
Quantitative real-time PCR was performed as detailed
described previously
8,10
. Briefly, total RNA from frozen
rat liver tissues was extracted with TRIzol reagent (Invi-
trogen Inc., Grand Island, NY) and reverse-transcribed to
cDNA (Thermo Scientific Revert Aid). The expressions of
the target genes were detected by SYBR Green quantita-
tive real-time polymerase chain reaction, with β-actin as
an internal control. The primer sequences used are listed
in Supplementary Table 1.
Western blot analysis
Western blot was performed as previously reported
44
.
Briefly, the membranes were incubated overnight at 4 °C
with the following corresponding primary antibodies: anti-
HECTD1 (1:200, Santa Cruz Biotechnology, Santa Cruz,
CA, USA), anti-HECTD2, anti-HECTD3 (1:800, BIOS,
Beijing, China), anti-TRAF3 (1:200, Affinity Biosciences,
Shanghai, China), anti-phospho-IKKβ(Tyr199), anti-
phospho-IκBα(Ser32/Ser36), anti-phospho-p65 (Ser536)
(1:500, Affinity Biosciences, Shanghai, China), anti-IKKβ,
anti-IκBα, anti-p65 (1:1000, Proteintech, Wuhan, China),
anti-GST tag, anti-His tag, anti-FLAG tag, anti-HA tag
(1:5000, Proteintech, Wuhan, China), and anti-β-actin
(1:3000, Proteintech, Wuhan, China) antibodies.
Co-IP and ubiquitination analysis
Total proteins were extracted from liver tissues and BRL-
3A cells as previously reported
44
. An equal amount of anti-
HECTD3 or anti-TRAF3 or anti-FLAG tag antibody was
added to 500 μg of protein and gently shaken at 4 °C over-
night. Immunocomplexes were acquired by adding 40 μLof
protein A +G agarose beads (Beyotime Institute of Bio-
technology, Shanghai, China). The mixtures were then
gently shaken at 4 °C for 4 h. The mixture was centrifuged at
1000 × gfor 5 min at 4 °C, and the supernatant was
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discarded. The sediment was washed five times using ice-
cold PBS. The immunocomplexes were boiled in sodium
dodecyl sulfate sample buffer for 5 min to separate them
from the beads. Then, the specimens were examined via
immunoblotting with anti-HECTD3 and anti-TRAF3 anti-
bodies or anti-FLAG tag antibody according to the manu-
facturer’s instructions. For ubiquitination analysis, the
immunocomplexes were analyzed by immunoblotting with
an anti-ubiquitin or anti-HA tag antibody.
Recombinant HECTD3 and TRAF3 expression and
purification
GST-tagged TRAF3, His-tagged full-length human
HECTD3, His-tagged DOC domain (amino acid residues
219–397) and HECT domain (amino acid residues
512–857) were cloned as a BamHI XhoI fragment into
pGEX-4T-1 or pet32a (Invitrogen, Shanghai, China) and
expressed as a fusion in Escherichia. Coli BL21 cells. The
cells were cultured at 37 °C with shaking until logarithmic
phase and isopropyl β-D-thiogalactoside (IPTG) was added
to a final concentration of 0.1 mM. Bacteria were collected
by centrifuging after 16 h shaking at 16 °C. GST-tagged
TRAF3 was purified using GSH Purose 4 Fast Flow beads
(Qianchun Bio, Yancheng, China) in 10 mM PBS (pH 8.0)
containing 10% glycerol and eluted using 50 mM Tris-HCl
(pH 8.0) containing 20 mM glutathione. The His-tagged
HECTD3, His-tagged DOC, and His-tagged HECT
domains were purified using Ni-NTA Purose 6 Fast Flow
beads (Qianchun Bio, Yancheng, China) in 10 mM PBS (pH
8.0) containing 10% glycerol and eluted with 10 mM PBS
(pH 8.0) containing 500 mM imidazole.
GST pull-down assay
GST and GST-TRAF3 were constructed using Escher-
ichia. Coli BL21 cells and were purified with GSH beads.
The His-tagged full-length HECTD3 fusion protein and
His-tagged DOC and HECT domains were expressed in
BL21 (DE3) cells, purified, and collected with Ni-NTA
beads. They were rotated with GST and GST-TRAF3 at
4 °C for 16 h and added to GSH Purose 4 Fast Flow beads
(Qianchun Bio, Yancheng, China) for an additional 4 h at
4 °C. After centrifugation and three washes, the beads were
eluted with 50 μL of 1× sodium dodecyl sulfate poly-
acrylamide gel electrophoresis (SDS-PAGE) loading buffer
and boiled for 5 min, followed by western blot.
Cell viability assay
The tetrazolium salt CCK-8 (Bimake, China) was used
to determine cell viability according to the manufacturer’s
protocol. BRL-3A cells were cultured in 96-well plates.
After pre-treatment, CCK-8 was added to the cell culture
medium at 1:10 and sequentially incubated at 37 °C for
1 h. Finally, absorbance at 450 nm was measured using a
microplate reader (Molecular Devices, CA, USA).
ROS staining
The ROS assay kit (Beyotime, Shanghai, China) was
used to quantify intracellular ROS levels. BRL-3A cells
were cultured in 24-well plates. After cell transfection and
modeling, BRL-3A cells were washed thrice with PBS.
Then, they were loaded with DCFH-DA (10 μM) for
20 min at 37 °C. To fully remove the superfluous DCFH-
DA probe, BRL-3A cells were washed thrice with PBS,
fixed in 4% paraformaldehyde for 20 min at room tem-
perature, and washed three times with PBS. The cells were
then observed and imaged using a fluorescence micro-
scope (Olympus, Tokyo, Japan).
TMA analysis
TMA was constructed from paraformaldehyde-fixed and
paraffin-embedded specimens. Briefly, core human liver
tissue specimens (diameter: 3 mm) were collected from
typical regions of the individual donor blocks and accurately
arrayed into the recipient paraffin block (45 × 22 mm) using
a custom-built instrument (Beecher Instruments, Silver
Spring, MD, USA). Then, the recipient paraffinblockwas
incubated at 62 °C for 1 h, and the surface of the block was
smoothed. Consecutive sections (5 μm) of the TMA block
were cut with a microtome. The TMA blocks were then
subjectedtoH&EandIHCstaining.
Human tissue specimens
Human liver samples were collected from the Zhongnan
Hospital of Wuhan University. (Wuhan, China). Ethical
approval was obtained from Medical Ethics Committee of
Zhongnan Hospital of Wuhan University (approval
number 2020122). Informed consent was obtained from
all participants.
Statistical analysis
All experimental data are shown as the mean ± standard
deviation. Groups were compared using Student’s two-
tailed unpaired t-test, one or two-way ANOVA followed
by Tukey’s post-hoc test. Data analysis was performed
using GraphPad Prism 8.0 (GraphPad Prism Software, La
Jolla, CA, USA). P< 0.05 was considered significant.
Acknowledgements
All authors declare that they have no acknowledgement.
Funding
This study was funded by the Medical Science Advancement Program (Clinical
Medicine) of Wuhan University (grant number TFLC2018003); the National
Natural Science Foundation of China (grant number 81700657); and the
Natural Science Foundation of Hubei Province (grant number 2016CFA094).
Author details
1
Zhongnan Hospital of Wuhan University, Institute of Hepatobiliary Diseases of
Wuhan University, Transplant Center of Wuhan University, Hubei Key
Laboratory of Medical Technology on Transplantation, Engineering Research
Center of Natural Polymer-based Medical Materials in Hubei Province, Wuhan,
Zhou et al. Cell Death and Disease (2021) 12:211 Page 15 of 17
Official journal of the Cell Death Differentiation Association
China.
2
The First Affiliated Hospital, Zhejiang University School of Medicine,
Department of Hepatobiliary and Pancreatic Surgery, Zhejiang Provincial Key
Laboratory of Pancreatic Disease, Innovation Center for the Study of Pancreatic
Diseases, Hangzhou, China.
3
The 3rd Xiangya Hospital of Central South
University, Research Center of National Health Ministry on Transplantation
Medicine Engineering and Technology, Changsha, China
Author contributions
W.Z., Z.B.Z., Z.Z.L., S.J.Y., and Q.F.Y. conceived and designed the study. W.Z. and
D.N.L. carried out the main experiments, analyzed the data, and drafted this
manuscript. Z.B.Z., Q.Y.Z., H.Y.X., and S.P. collected the human liver tissue
specimens. A.X.L., Z.S.L., and Y.F.W. gave advice and assisted in the
experiments. All authors reviewed and approved the manuscript.
Conflict of interest
The authors declare no competing interests.
Ethics statement
The study was approved by Medical Ethics Committee of Zhongnan Hospital
of Wuhan University.
Informed consent
All patients provided written informed consent.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Supplementary information The online version contains supplementary
material available at https://doi.org/10.1038/s41419-021-03493-2.
Received: 14 December 2020 Revised: 30 January 2021 Accepted: 1
February 2021
References
1. Kron,P.,Schlegel,A.,Mancina,L.,Clavien,P.A.&Dutkowski,P.Hypothermic
oxygenated perfusion (HOPE) for fatty liver grafts in rats and humans. J.
Hepatol. 68,82–91 (2018).
2. Monbaliu, D., Pirenne, J. & Talbot, D. Liver transplantation using donation after
cardiac death donors. J. Hepatol. 56, 474–485 (2012).
3. Compagnon, P. et al. An oxygenated and transportable machine perfusion
system fully rescues liver grafts exposed to lethal ischemic damage in a pig
model of DCD liver transplantation. Transplantation 101, e205–e213 (2017).
4. deVera,M.E.etal.Livertransplantation using donation after cardiac death
donors: long-term follow-up from a single center. Am.J.Transpl.9,773–781
(2009).
5. Liu, Z. et al. Pretreatment donors after circulatory death with simvastatin
alleviates liver ischemia reperfusion injury through a KLF2-dependent
mechanism in rat. Oxid.Med.CellLongev.2017, 3861914 (2017).
6. Liu, Y. et al. Activation of YAP attenuates hepatic damage and fibrosis in liver
ischemia-reperfusion injury. J. Hepatol. 71,719–730 (2019).
7. Sosa,R.A.,etal.Disulfide high-mobility group box 1 drives ischemia-
reperfusion injury in human liver transplantation. Hepatology https://doi.org/
10.1002/hep.31324 (2020).
8. He, W. et al. Hypothermic oxygenated perfusion (HOPE) attenuates ischemia/
reperfusion injury in the liver through inhibition of the TXNIP/NLRP3in-
flammasome pathway in a rat model of donation after cardiac death. FASEB J.
32, 6212–6227 (2018).
9. Schlegel, A., de Rougemont, O., Graf, R., Clavien, P. A. & Dutkowski, P. Protective
mechanisms of end-ischemic cold machine perfusion in DCD liver grafts. J.
Hepatol. 58,278–286 (2013).
10. Zeng, X. et al. Hypothermic oxygenated machine perfusion alleviates donation
after circulatory death liver injury through regulating p-selectin-dependent
and -independent pathways in mice. Transplantation 103,918–928 (2019).
11. Li, F. et al. HECTD3 mediates TRAF3 polyubiquitination and type I interferon
induction during bacterial infection. J. Clin. Invest. 128,4148–4162 (2018).
12. Rotin,D.&Kumar,S.Physiologicalfunctions of the HECT family of ubiquitin
ligases. Nat. Rev. Mol. Cell Biol. 10,398–409 (2009).
13. Li, Y. et al. The HECTD3 E3 ubiquitin ligase facilitates cancer cell survival by
promotingK63-linkedpolyubiquitination of caspase-8. Cell Death Dis. 4,e935
(2013).
14. Jiang,Q.,Li,F.,Cheng,Z.,Kong,Y.& Chen, C. The role of E3 ubiquitin ligase
HECTD3 in cancer and beyond. Cell Mol. Life Sci. 77,1483–1495 (2020).
15. Kathania, M. et al. Itch inhibits IL-17-mediated colon inflammation and
tumorigenesis by ROR-gammat ubiquitination. Nat. Immunol. 17,997–1004
(2016).
16. Cho,J.J.etal.Hectd3promotespathogenic Th17 lineage through Stat3
activation and Malt1 signaling in neuroinflammation. Nat. Commun. 10,701
(2019).
17. Arkee,T.&Bishop,G.A.TRAFfamilymoleculesinTcells:multiplereceptors
and functions. J. Leukoc. Biol. 107,907–915 (2020).
18. Hu, J. et al. Targeting TRAF3 signaling protects against hepatic ischemia/
reperfusions injury. J. Hepatol. 64,146–159 (2016).
19. Wallis, A. M. & Bishop, G. A. TRAF3 regulation of inhibitory signaling pathways
in B and T lymphocytes by kinase and phosphatase localization. J. Leukoc. Biol.
https://doi.org/10.1002/JLB.2MIR0817-339RR (2018).
20. Fochi, S. et al. TRAF3 is required for NF-kappaB pathway activation mediated
by HTLV tax proteins. Front. Microbiol. 10, 1302 (2019).
21. Perez-Chacon, G. et al. DysregulatedTRAF3andBCL2expressionpromotes
multiple classes of mature non-hodgkin B cell lymphoma in mice. Front.
Immunol. 9, 3114 (2018).
22. Lee,S.C.,Kim,K.H.,Kim,O.H.,Lee,S.K.&Kim,S.J.Activationofautophagyby
everolimus confers hepatoprotection against ischemia-reperfusion injury. Am.
J. Transpl. 16, 2042–2054 (2016).
23. Zhang, L. et al. Prolonged warm ischemia aggravates hepatic mitochondria
damage and apoptosis in DCD liver by regulating Ca(2+)/CaM/CaMKII sig-
naling pathway. Int. J. Clin. Exp. Pathol. 12,217–228 (2019).
24. Guixe-Muntet, S. et al. Cross-talk between autophagy and KLF2 determines
endothelial cell phenotype and microvascular function in acute liver injury. J.
Hepatol. 66,86–94 (2017).
25. Stolz, D. B. et al. Sinusoidal endothelial cell repopulation following ischemia/
reperfusion injury in rat liver transplantation. Hepatology 46,1464
–1475 (2007).
26. Ford, A. J., Jain, G. & Rajagopalan, P. Designing a fibrotic microenvironment to
investigate changes in human liver sinusoidal endothelial cell function. Acta
Biomater. 24,220–227 (2015).
27. Sorensen, K. K., Simon-Santamaria, J., McCuskey, R. S. & Smedsrod, B. Liver
sinusoidal endothelial cells. Compr. Physiol. 5,1751–1774 (2015).
28. Zhang,Y.Y.etal.CD31regulatesmetastasis by inducing epithelial-
mesenchymal transition in hepatocellular carcinoma via the ITGB1-FAK-Akt
signaling pathway. Cancer Lett. 429,29–40 (2018).
29. Li, Y. et al. The HECTD3 E3 ubiquitin ligase suppresses cisplatin-induced
apoptosis via stabilizing MALT1. Neoplasia 15,39–48 (2013).
30. Li,Z.,Zhou,L.,Prodromou,C.,Savic,V.&Pearl,L.H.HECTD3mediatesan
HSP90-dependent degradation pathway for protein kinase clients. Cell Rep. 19,
2515–2528 (2017).
31. Shu,T.,Li,Y.,Wu,X.,Li,B.&Liu,Z.Down-regulationofHECTD3byHER2
inhibition makes serous ovarian cancer cells sensitive to platinum treatment.
Cancer Lett. 411,65–73 (2017).
32. Zohn, I. E., Anderson, K. V. & Niswander, L. The Hectd1 ubiquitin ligase is
required for development of the head mesenchyme and neural tube closure.
Dev. Biol. 306,208–221 (2007).
33. Scheffner, M. & Staub, O. HECT E3s and human disease. BMC Biochem. 8,S6
(2007).
34. Heaton, S. M., Borg, N. A. & Dixit, V. M. Ubiquitin in the activation and
attenuation of innate antiviral immunity. J. Exp. Med. 213,1–13 (2016).
35. Tao,J.,Zhou,X.&Jiang,Z.cGAS-cGAMP-STING:thethreemusketeersof
cytosolic DNA sensing and signaling. IUBMB Life 68,858–870 (2016).
36. Zindel, J. & Kubes, P. DAMPs, PAMPs, and LAMPs in immunity and sterile
inflammation. Annu. Rev. Pathol. 15,493–518 (2020).
37. Miao,Y.,Wu,J.&Abraham,S.N.Ubiquitination of innate immune regulator
TRAF3 orchestrates expulsion of intracellular bacteria by exocyst complex.
Immunity 45,94–105 (2016).
38. Zhu, Q. et al. TRIM24 facilitates antiviral immunity through mediating
K63-linked TRAF3 ubiquitination. J. Exp. Med. 217, e20192083 (2020).
39. Rayego-Mateos, S. et al. TRAF3 modulation: novel mechanism for the anti-
inflammatory effects of the vitamin D receptor agonist paricalcitol in renal
disease. J. Am. Soc. Nephrol. 31,2026–2042 (2020).
Zhou et al. Cell Death and Disease (2021) 12:211 Page 16 of 17
Official journal of the Cell Death Differentiation Association
40. Huai, W. et al. Mint3 potentiates TLR3/4- and RIG-I-induced IFN-beta expres-
sion and antiviral immune responses. Proc. Natl Acad. Sci. USA 113,
11925–11930 (2016).
41. Schlegel,A.,Kron,P.,Graf,R.,Dutkowski,P.&Clavien,P.A.Warmvs.cold
perfusion techniques to rescue rodent liver grafts. J. Hepatol. 61, 1267–1275
(2014).
42. Wang, W. et al. Mild hypothermia pretreatment attenuates liver ischemia
reperfusion injury through inhibiting c-Jun NH2-terminal kinase phosphor-
ylation in rats. Transpl. Proc. 50, 259–266 (2018).
43. Suzuki, S., Toledo-Pereyra, L. H., Rodriguez, F. J. & Cejalvo, D. Neutrophil infil-
tration as an important factor in liver ischemia and reperfusion injury. Mod-
ulating effects of FK506 and cyclosporine. Transplantation 55,1265–1272
(1993).
44. Zhou, W. et al. PKCzeta phosphorylates TRAF2 to protect against intestinal
ischemia-reperfusion-induced injury. Cell Death Dis. 8, e2935 (2017).
45. Lin, D. et al. Aldehyde dehydrogenase 2 regulates autophagy via the Akt-
mTOR pathway to mitigate renal ischemia-reperfusion injury in hypothermic
machine perfusion. Life Sci. 253, 117705 (2020).
Zhou et al. Cell Death and Disease (2021) 12:211 Page 17 of 17
Official journal of the Cell Death Differentiation Association
