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The ER-localised Hrd1 ubiquitinates and inactivates Usp15 to promote 1
TLR4-induced inflammation during bacterial infection 2
Yao Lu1*, Ying Qiu1*, Peng Chen2, Haishuang Chang3, Luqiang Guo3, Fang Zhang4, Li 3
Ma1, Chi Zhang1, Xin Zheng1, Jun Xiao1, Ruiyue Zhong1, Lei Han1, Xiaoyan Xu1,5, 4
Yanbo Zhang1,6, Dangsheng Li1, Guisheng Zhong7, Rosemary Boyton8, Ying Huang3, 5
Yongning He3, Ronggui Hu2#, Bin Wei4,9#, Hongyan Wang1,10# 6
1 State Key Laboratory of Cell Biology, Key Laboratory of Systems Biology, CAS 7
Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and 8
Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, 9
Innovation Center for Cell Signaling Network, Shanghai, 200031, China; 2 State Key 10
Laboratory of Molecule Biology, Key Laboratory of Systems Biology, CAS Center for 11
Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell 12
Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, 13
Shanghai, 200031, China; 3 State Key Laboratory of Molecular Biology, National Center 14
for Protein Science Shanghai, Shanghai Science Research Center, Shanghai Key 15
Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell 16
Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of 17
Sciences, University of Chinese Academy of Sciences, Shanghai, 201210; 4 Wuhan 18
Institute of Virology, Chinese Academy of Sciences, Wuhan, China; 5 Experimental 19
Immunology Branch, National Cancer Institute, US National Institutes of Health, 20
Bethesda, Maryland, USA; 6 Division of Immunology, Department of Microbiology and 21
Immunobiology, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, 22
USA; 7 iHuman Institute, School of Life Science and Technology, ShanghaiTech 23
University, Shanghai, China; 8 Lung Immunology Group, Department of Infectious 24
Diseases, Faculty of Medicine, Imperial College London, London W12 0NN, United 25
Kingdom; 9 School of Life Sciences, Shanghai University, Shanghai 200444; 10 Cancer 26
Center, Shanghai Tenth People's Hospital, Tongji University, School of Medicine, 27
Shanghai 200072, China 28
29
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*Contributed equally; #Corresponding author: [email protected] ; 30
[email protected] ; [email protected] 31
Running title: Hrd1 enhances TLR4 pathway upon infection 32
Keywords: Hrd1, Ubiquitination, Usp15, TLR4 pathway, Inflammation and Infection 33
34
ABSTRACT 35
The special organelle-located MAVS, STING and TLR3 are important for clearing viral 36
infections. Although TLR4 triggers NF-κB activation to produce proinflammatory 37
cytokines for bacteria clearance, effectors with special organelle localisation have not 38
been identified. Here, we screened over 280 E3 ubiquitin ligases and discovered that the 39
endoplasmic reticulum-located Hrd1 regulated TLR4-induced inflammation during 40
bacterial infection. Hrd1 directly interacted with the deubiquitinating enzyme (DUB) 41
Usp15. Unlike the classical function of Hrd1 in ER-associated degradation, Usp15 was 42
not degraded but lost its DUB activity for IκBα deubiquitination, resulting in excessive 43
NF-κB activation. Importantly, Hrd1 deficiency in macrophages protected mice against 44
LPS-induced septic shock, and knock-down of Usp15 in Hrd1 KO macrophages restored 45
the reduced IL-6 production. This study has proposed the crosstalk between Hrd1 and 46
TLR4 linking the ER-plasma membrane function during bacterial infection. 47
48
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Introduction 49
Macrophages express pattern recognition receptors (PRRs) such as Toll-like 50
receptors (TLRs) and RIG-like receptors (RLRs) to sense pathogen-associated molecular 51
patterns (PAMPs) and trigger the innate immune response, leading to inflammation and 52
microbe clearance.1 The mitochondria-located MAVS (Mitochondrial antiviral-signaling 53
protein, also named VISA (virus-induced signaling adapter), IPS-1 and Cardif)2, 3, 4, 5, the 54
endoplasmic reticulum (ER)-located STING (Stimulator of interferon genes)6, 7, 8 and the 55
endosome-located TLR3 (Toll-like receptor 3)9 are important for type I IFN production 56
and clearance of viral infections4. Although the surface receptor TLR4-triggered NF-κB 57
activation is well studied for proinflammatory cytokine production and bacteria clearance, 58
downstream effectors with special organelle localisation have not been identified in the 59
TLR4 pathway. In recent years, studies of membrane contact sites within cells and their 60
role have been rapidly advancing, in particular insights have recently demonstrated the 61
contact sites exist and function between the largest organelle endoplasmic reticulum (ER) 62
and other organelles for cell homeostasis or disease pathogenesis by sensing the intra- or 63
extracellular stimulation 2, 3, 6, 10, 11. 64
While a balanced inflammatory response is pivotal to protecting the host against 65
microbes and self-injury, excessive activation of the TLR or RLR signalling pathway 66
could lead to serious inflammatory diseases, including septic shock or autoimmune 67
diseases. Septic shock is the most common cause of death in hospitalized patients and the 68
proinflammatory cytokine IL-6 is crucial in the pathophysiology of severe sepsis, and 69
IL-6 levels most significantly correlate with mortality rates compared to other cytokines12. 70
Accumulating studies have shown that E3 ubiquitin ligases are involved in TLR 71
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signalling. TRAF6 (TNF receptor associated factor 6), a typical RING-type E3, can be 72
autoubiquitinated at Lys124, which is then recognized by the TAB (TGF-β activated 73
kinase 1 binding protein) 2/3 complex to further activate TAK1 (TGF-β activated kinase 74
1) and NEMO (NF-κB essential modulator). Pellino-1 increases LPS-driven Lys63-linked 75
polyubiquitination of IRAK1, TBK1 (TANK binding kinase 1) and TAK1 in TLR 76
signalling. Since E3 ligases and their substrates can be targeted to attenuate excessive 77
inflammation and sepsis, we were interested in investigating whether any E3 ligases with 78
special organelle localisation could be identified in the TLR4 pathway. 79
We screened over 280 E3 ligases using Dharmacon RNAi Screening Libraries and 80
identified Hrd1 as a RING-type E3 ubiquitin ligase, which positively regulated IL-6 81
production in LPS-treated macrophages. Hrd1, a homologue of yeast Hrd1p/Der3p13, 82
contains a transmembrane domain and is specifically located in the endoplasmic 83
reticulum (ER). The best-defined function of Hrd1 is to ubiquitinate misfolded/unfolded 84
proteins with help from other proteins in the ER-associated degradation (ERAD) 85
complex14, 15, 16, 17, which protects cells from ER-stress-induced apoptosis18. In agreement 86
with this role, Hrd1 (also known as Synoviolin, Syvn1) expression is enhanced in 87
synovial fibroblasts from rheumatoid arthritis (RA) patients19, and Hrd1+/− mice are 88
resistant to collagen-induced arthritis due to increased synovial cell apoptosis20, 21. 89
Previous studies have demonstrated that TLR4 is highly expressed in RA synovial tissue 90
lining and sublining macrophages22, and excessive levels of TNF-α and IL-6 accelerate 91
RA development. However, no knowledge is available about how Hrd1 affects 92
TLR4-induced inflammation. More importantly, the ER has a broad localisation 93
throughout the cell and can form direct physically contacts with the cell membrane11, 23. 94
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We found that macrophages enhance ER membranes upon bacterial infection. Therefore, 95
it was interesting to further elucidate how the ER-located Hrd1 participates in 96
TLR4-induced inflammation in macrophages during bacterial infection. 97
This study has identified an ERAD-independent function of Hrd1 to increase 98
TLR4-induced proinflammatory cytokine production. We fished out Usp15 99
(Ubiquitin-specific protease 15) as a binding partner for Hrd1. Usp15 is a member of the 100
largest subfamily of cysteine protease DUBs (deubiquitinating enzymes). Prior studies 101
have indicated that Usp15 promotes cell survival by stabilizing IκBα in TNF-α stimulated 102
HeLa cells24, and Usp15 promotes type I interferon responses and pathogenesis during 103
neuroinflammation25. Other studies have demonstrated Usp15 function in anti-tumor 104
response, including that Usp15 regulates p53 function to promote cancer-cell survival and 105
Usp15 inhibits T cell activation and immune surveillance26. However, it remains unclear 106
about how Usp15 affects TLR4-induced inflammation. This study has demonstrated that 107
Hrd1 promoted polyubiquitination of Lys21 in Usp15. Unlike other Hrd1 substrates, 108
ubiquitinated Usp15 was not degraded, but rather lost its DUB activity and failed to 109
deubiquitinate IκBα, which resulted in excessive TLR4-NF-κB activation. 110
Results 111
RNAi screening identifies the ER-localised Hrd1 to positively regulate inflammation 112
in LPS-stimulated macrophages 113
To identify E3 ligases involved in the regulation of TLR4-signalling, we transfected 114
over 280 siRNAs (Dharmacon RNAi Screening Libraries) into murine primary peritoneal 115
exudate macrophages (PEMs), followed by LPS stimulation for 6 hr. IL-6 concentrations 116
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in supernatants were measured by ELISA (Figure S1a).We controlled macrophage 117
survival by checking the am-blue absorbance and excluded genes that significantly 118
induced macrophage death (indicated by blue colour in Figure 1a). Approximately 20 119
candidate genes were identified to affect IL-6 production, including Birc2 and Traf5 that 120
were previously identified in regulating inflammation (Figure 1a)27, 28. We next verified 121
the functions of several candidate genes, including Hrd1, using single siRNAs in smart 122
pools. In order to select our most interested genes for further investigation, we treated 123
PEMs with LPS-coated latex beads. Interestingly, electron microscopy analysis showed 124
that macrophages displayed enhanced ER membrane upon stimulated with LPS-coated 125
latex beads (Figures 1b and S1b). We next checked how ER might be affected in 126
response to bacterial infection in MEFs, which were transiently transfected with 127
KDEL-mCherry to label ER. After infected with Salmonella typhimurium (strain 128
SL1344), mCherry formed aggregation in response to bacterial infection (Figure 1c). 129
ER is the largest organelle in the cell which forms an interconnected network with 130
almost every membrane-bound organelles, including cell membrane and mitochondria29, 131
30. Advanced studies have suggested that ER is essential for cell homeostasis or disease 132
pathogenesis by sensing the intra- or extracellular stimulation through contact sites with 133
other organelles.31 Despite that Hepatitis B virus (HBV) might induce ER dysfunctions 134
that leads to liver injury32; and the ER-located STING participates in the innate immune 135
response against viruses7, little is known about how ER functions in bacterial infection 136
and no many effectors with ER localisation have not been identified in the TLR4 pathway. 137
We therefore took this advantage to further elucidate how the ER-located Hrd1 138
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cooperates with the cell membrane receptor TLR4 to regulate inflammation in vivo and in 139
vitro. 140
Knock-down (KD) of Hrd1 using 4 different siRNAs reproducibly reduced the 141
mRNA levels of Il6 and Tnfa in LPS-stimulated PEMs (Figure 1d, KD efficiencies of the 142
4 different siHrd1 were shown in Figure S1c). Similarly, when infected with 143
Gram-negative bacteria, such as Salmonella typhimurium (strain SL1344) and 144
Escherichia coli (E. coli), Hrd1-silenced macrophages produced much lower levels of Il6 145
and Tnfa (Figure 1e). We also confirmed decreased concentrations of IL-6 and TNF-α by 146
ELISA in Hrd1-silenced PEMs after LPS stimulation (Figure 1f, siHrd1 KD efficiency is 147
shown in Figure S1d.). In agreement with these results, stable overexpression of Hrd1 in 148
human THP-1 cells or primary mouse embryonic fibroblasts (MEFs) significantly 149
increased LPS-induced Il6 production at the mRNA level (Figure 1g). 150
We then performed the luciferase reporter assay to determine how Hrd1 cooperates 151
with MyD88-dependent and -independent pathways to promote TLR4-NF-κB activation. 152
The key TLR4 signalling effectors were transfected alone or together with Hrd1 into 153
HEK293T cells to measure luciferase readings and the ER location of overexpressed 154
Hrd1 was determined by Western blot (Figure S1e). Hrd1 co-expression cooperated with 155
MyD88, TRAF6, IKKα/β (Inhibitor of nuclear factor kappa B kinases alpha/beta) and 156
TRIF (Toll/IL-1 receptor domain-containing adaptor) to substantially further increase 157
luciferase readings (Figure 1h, middle and right panels) compared to the effect mediated 158
by transfecting the TLR4 signalling effectors alone. Furthermore, this enhancement was 159
shown to be Hrd1 dose-dependent when either TRAF6 or TRIF was co-transfected 160
(Figure 1i, Hrd1 levels were shown in Figure S1f). In contrast, Hrd1 did not further 161
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increase luciferase readings when it was co-expressed with p65 in HEK293T cells 162
(Figure 1h), suggesting that Hrd1 was located upstream of p65 and downstream of 163
IKKα/β. We also noted that overexpression of Hrd1 alone slightly enhanced luciferase 164
readings (Figure 1h, left panel), but this effect was very minor compared to those 165
mediated by transfecting MyD88, TRAF6, IKKα, IKKβ, TRIF or p65 alone (Figure 1h, 166
white columns). 167
To elucidate whether the ER-localisation of Hrd1 was critical for NF-κB activation, 168
we truncated the transmembrane domain of Hrd1, termed ΔTM (Figure 1j, left and lower 169
panel). Using the NF-κB luciferase system, we indeed found that the Hrd1 ΔTM mutant 170
failed to increase luciferase readings compared to WT Hrd1 (Figure 1j, left and top panel). 171
To better explore the importance of the ER location of Hrd1 in the regulation of 172
inflammation, Hrd1 was targeted to mitochondrial membrane (termed mito-Hrd1) (Figure 173
S1g & Figure 1j, right and lower panel). Similar with the ΔTM truncation, mito-Hrd1 174
failed to increase NF-κB luciferase readings compared to WT Hrd1 (Figure 1j, right panel) 175
and could not promote pro-inflammatory cytokines expression in MEFs (Figure 1k, the 176
expression level of Hrd1 was shown in right panel). These results taken together, we have 177
identified that the ER-located E3 ligase Hrd1 enhances TLR4-induced inflammation in 178
macrophages. 179
180
Hrd1 KO macrophages reduce TLR4-induced inflammation and NF-κB activation 181
To better understand Hrd1 function, we generated Hrd1 conditional knock-out mice. 182
Exon 6 in the Hrd1 gene was floxed, resulting in a stop codon after 125 amino acids 183
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(named Hrd1fl/fl, Figure S2a). Hrd1fl/fl mice were crossed with Lysozyme M (LysM)-Cre 184
mice to generate conditional knock-out (cKO) mice lacking Hrd1 expression in myeloid 185
cells. The efficiency of Hrd1 KO in macrophages was confirmed by western blot (Figure 186
2a). Hrd1 deficiency did not affect the development of bone marrow cell-derived 187
macrophage, which showed normal mRNA expression levels of Adgre1 (Adhesion G 188
Protein-Coupled Receptor E1, also known as F4/80) and MerTK (Mer tyrosine kinase) as 189
well as normal percentages of F4/80+CD11b+ macrophages (Figure S2b). In addition, 190
when bred in an SPF facility, Hrd1 cKO mice showed normal T cell development in the 191
thymus and normal percentages of B220+, CD4+, CD8+ lymphocytes and F4/80+, CD11b+ 192
macrophages in the spleen (Figure S2c). Although Hrd1 is associated with 193
ER-stress-induced cell apoptosis20, Hrd1-deficient macrophages did not exhibit reduced 194
cell viability after being challenged by E. coli infection (Figure S2d). Also Hrd1 195
deficiency did not modulate LPS- and ATP-induced cleavage of Caspase1 (Figure S2e). 196
This result was in agreement with the unchanged cell survival we observed after 197
knock-down of Hrd1 in our siRNA library screening system (Figure 1a). 198
Consistent with our luciferase data, Hrd1-deficient PEMs exhibited no differences in 199
IKKα/β phosphorylation (Figure 2b, left panel). Upon IKKα/β phosphorylation and 200
activation, IκBα is phosphorylated then polyubiquitinated for subsequent degradation; 201
this results in nuclear entry of NF-κB to turn on target gene expression33. Indeed, we 202
observed that Hrd1-deficient macrophages degraded IκBα less efficiently (Figure 2b, 203
right panel) and less p65 translocation into the nucleus decreased (Figure 2c) upon LPS 204
stimulation. Knock-out or knock-down of Hrd1 in PEMs did not change phosphorylation 205
levels of JNK, ERK or p38 (Figures 2d and S2f). Similar to the data from our Hrd1 206
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knock-down experiments, Hrd1-deficient PEMs and bone marrow derived macrophages 207
(BMMs) also exhibited reduced expression of Il6, Tnfa and Il1b at the mRNA levels 208
(Figures 2e, Figures S2g) or concentrations of IL-6 and TNF-α (Figure 2f) after treatment 209
with LPS, Salmonella typhimurium or E. coli. To determine whether NF-κB is the major 210
transcription factor downstream of Hrd1, we next blocked NF-κB activation by 211
knock-down of the NF-κB subunit p65 or by using the inhibitor PDTC (pyrrolidine 212
dithiocarbamate) (Figures 2g, S2i, sip65 KD efficiency is shown in Figure S2h). 213
Knock-down of p65 (Figure 2g) and PDTC treatment (Figure S2i) both profoundly 214
suppressed IL-6 expression to similar levels in WT and Hrd1 KO macrophages. 215
Interestingly, unlike the LPS/TLR4-induced response, Hrd1 did not modulate CpG 216
(TLR9 stimuli)-induced IL-6 production, Poly(I:C) (TLR3 stimuli)-induced IFN-β 217
production (Figure 2h) and PGN (TLR2 stimuli)-induced pro-inflammatory cytokine 218
production (Figure S2j). Collectively, we have demonstrated that Hrd1 specifically 219
regulates TLR4-induced IκBα degradation to increase NF-κB activation and 220
proinflammatory cytokine production in macrophages, without significantly affecting 221
TLR2/TLR3/TLR9 signalling. 222
223
The E3 ligase activity of Hrd1 is critical for TLR4-induced NF-κB activation 224
independent of ERAD 225
As an ER-located E3 ligase, the classical physiological function of Hrd1 is to 226
catalyse addition of ubiquitin molecules to lysine (Lys) residues in substrate proteins to 227
promote their degradation. After we confirmed that the ER location of Hrd1 is critical for 228
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TLR4 signalling (Figure 1j), we next assessed whether the E3 ligase activity of Hrd1 was 229
also required for modulating TLR4 signalling. Hrd1 contains 8 conserved cysteine and 230
histidine residues in the core of its RING domain that maintain its three-dimensional 231
structure. Based on previous reports, the cysteine residue at position 329 is critical, and 232
its mutation to serine (C329S) abolishes Hrd1 E3 activity18. Notably, the catalytic 233
inactive mutant C329S failed to elevate NF-κB luciferase activity when co-expressed 234
with MyD88, TRAF6 or TRIF (Figure 3a, similar overexpression levels of Hrd1 and 235
C329S were shown in the right panel). In agreement with this result, stable 236
overexpression of the C329S mutant in THP-1 cells (Figure S3a) decreased Lys48-linked 237
polyubiquitination of IκBα when compared to that seen in THP1 cells stable 238
overexpressing Hrd1 (Figure 3b). Stable overexpression of the C329S mutant or the 239
mitochondrial located Hrd1 mutant (i.e. mito-Hrd1) in MEFs could not promote 240
LPS-triggered IκBα degradation as effectively as those in MEFs expressing WT Hrd1 241
(Figure 3c). The expression levels of Hrd1 and its mutant were shown in Figure S3b and 242
Figure 1k, right panel. We performed immunoprecipitation experiments and found that 243
Hrd1 or the C329S mutant did not bind IκBα (Figure S3c), which does not support the 244
possibility that Hrd1 might directly ubiquitinate IκBα for degradation. Also Hrd1 or the 245
C329S mutant could not interact with TLR4, Myd88, TRIF or TBK1 (Figure S3c, left 246
panel), which indeed bound Hrd3 (Figure S3c, right panel). We next purified the nucleus 247
and cytoplasm fractions, and the C329S mutant reduced p65 levels in the nuclei of THP-1 248
cells (Figure 3d). Using immunofluorescence strategies, we also confirmed that stable 249
overexpression of the C329S mutant decreased p65 translocation into the nucleus in 250
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MEFs (Figure 3e, the relative amount of p65 in the nucleus was quantified in the right 251
panel). 252
Importantly, we reconstituted Hrd1-deficient BMMs to stably overexpress WT Hrd1, 253
C329S, ΔTM mutant or a GFP control using a retrovirus transduction system and similar 254
overexpression levels were confirmed by FACS analysis (Figure S3d, about 20% cells 255
were successfully transduced). Partially reconstituted WT Hrd1 expression, but not the 256
catalytic inactive mutant C329S or the transmembrane domain deletion mutant ΔTM, 257
could rescue IL-6 production in some degree in Hrd1-deficient BMMs (Figure 3f). 258
Hrd1, a core component in regulating ERAD14, 34, was recently elucidated to form a 259
dimer with Hrd3 (also named Sel1L) during ERAD35. To determine whether Hrd1- or 260
Hrd3-dependent ERAD was involved in TLR4-signalling, we used two siRNAs to knock 261
down Hrd3 expression in PEMs (Figure S3e). Knock-down of Hrd3 (Figure S3e) or Hrd1 262
KO PEMs (Figure 3g) significantly promoted tunicamycin-induced expression of 263
unfolded protein response (UPR)-target genes such as ERdj4 (Dnajb9), Bip (Hspa5), 264
CHOP (Ddit3) and the splicing of Xbp1.Unexpectedly, knock-down of Hrd3 for 36 hr in 265
PEMs (Hrd1 expression was not affected at this time point: Figure S3f, right panel) did 266
not significantly affect LPS-induced Il6 production at the mRNA level (Figure S3f). We 267
further investigated whether Hrd1 deficiency affects expression of ER stress-associated 268
proteins upon triggering TLR4 signalling. In agreement with other reports36, we observed 269
that comparing to tunicamycin treatment, both LPS stimulation and Gram-negative 270
bacteria infection minimally affected or even reduced the ER stress response in WT 271
control cells (Figure 3g and Figure S3g). In addtion, in response to LPS or bacteria 272
treatment, Hrd1-deficient (Figure 3g) or Hrd1 knock-down (Figure S3g) macrophages did 273
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not exhibit significantly altered expression levels of ER stress-associated genes. Together, 274
we have elucidated that Hrd1 depends on its E3 ligase catalytic activity and its ER 275
location to accelerate TLR4-induced IκBα degradation and p65 translocation, which 276
might be independent of ERAD. 277
278
The ER-localised Hrd1 directly binds Usp15 and regulates TLR4-induced 279
inflammation 280
Because Hrd1’s E3 ligase activity is critical for TLR4-NF-κB activation, we next 281
needed to determine its key substrate. To answer this question, human THP-1 cells stably 282
expressing Flag-tagged Hrd1 were stimulated with LPS, after which immunoprecipitation 283
using anti-Flag antibodies was performed for further mass spectrometry (MS) analysis. In 284
addition to proteins previously reported to participate in ERAD (i.e. Hrd3), our MS 285
analysis also identified interesting candidates including kinases, helicases and adaptor 286
proteins. We confirmed by immunoprecipitation assay that some candidate such as 287
WDR1 indeed interacted with Hrd1 in HEK293T cells, which however did not affect 288
NF-κB activation in the luciferase reporter assay (Figure S4a). After initial screening and 289
validation, we identified Usp15 (Ubiquitin specific peptidase 15) as an interesting 290
candidate for three reasons: First, Usp15 is a classical deubiquitinating enzyme (DUB) 291
and only a few studies to date have reported a DUB itself to bind and be ubiquitinated by 292
an E3 ligase. Second, Usp15 showed inhibitory effect on NF-κB activation when 293
co-transfected with MyD88 in HEK293T cells (Figure S4a). Third, Usp15 was previously 294
suggested to deubiquitinate IκBα in TNF-α-stimulated HeLa cells24, and, interestingly, 295
we observed a link between Hrd1 E3 ligase activity and IκBα degradation upon LPS 296
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treatment in macrophages (Figures 2b and 3c). We therefore investigated whether Usp15 297
is the downstream effector through which the ER-located Hrd1 regulates IκBα 298
degradation. 299
To confirm the interaction between Hrd1 and Usp15, a yeast two-hybrid system was 300
used. With the positive and negative controls, yeast indeed formed clones on the yeast 301
SD-Leu-Trp-His-Ura (SD-4) selection media after transfection with pDEST32-Hrd1 and 302
pDEST22-Usp15 plasmids (Figure 4a). To examine their direct interaction, plasmid 303
which expressed GST-tagged WT Hrd1 or the transmembrane domain truncation mutant 304
(i.e., ΔTM) as well as His-tagged Usp15 was respectively transformed into the bacterial 305
strain BL21 followed by protein purification. Using an in vitro pull-down assay, we 306
confirmed that WT Hrd1, but not Hrd1ΔTM, directly bound Usp15 (Figure 4b). 307
We next examined endogenous interactions between Hrd1 and Usp15 and detected 308
endogenous Hrd1 protein in anti-Usp15 immunoprecipitates from both PEMs (Figure 4c) 309
and primary MEFs (Figure S4b). Importantly, this endogenous interaction appeared to be 310
enhanced by LPS stimulation (Figures 4c and S4b). Interestingly, we could detect the 311
formation of endogenous protein complex containing Hrd1, Hrd3 and Usp15 in 312
anti-Usp15 immunoprecipitation. However, anti-Usp15 immunoprecipitation failed to 313
pull down Hrd3 and Hrd1 in Hrd1 KO PEMs (Figure S4c). This suggests that Usp15 314
might not be able to bind Hrd3 directly. Next, MEFs were transfected with Flag-tagged 315
Hrd1 and immunostained with anti-Flag and anti-Usp15 antibodies. Although Usp15 was 316
indeed located in the nucleus, we also observed interaction between Hrd1 and Usp15 in 317
the cytoplasm (Figure S4d). To further confirm these results, a cellular fractionation 318
assay was performed in lysed PEMs using centrifugation to harvest microsomes from 319
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fragmented ER. Calnexin was used as a positive control to identify ER sediment, while 320
Caspase3 and Aif, which do not appear in the ER, were used as negative controls. In 321
agreement with other reports, we detected both Hrd1 and Usp15 in ER sediments37. 322
Interestingly, more Usp15 was translocated into the ER after LPS stimulation (Figure 4d). 323
Immunofluorescence assays also showed that Usp15 colocalised with the ER marker 324
Calnexin, and this colocalisation was enhanced by LPS treatment (Figure S4e, Pearson’s 325
correlation value was increased from 0.58 to 0.68). 326
We next performed a domain mapping experiment and used different tags to 327
precipitate WT Hrd1 and the transmembrane domain truncation mutant (ΔTM) as well as 328
Usp15. HA-tagged Usp15 could co-precipitate Flag-tagged Hrd1 in HEK293T cells 329
(Figure S4f). We also used Myc-tagged Hrd1 and Flag-tagged Usp15 to repeat the 330
immunoprecipitation assay and confirmed their interaction (Figure S4g). Next, 331
Flag-tagged WT Hrd1 or ΔTM was co-expressed with HA-tagged Usp15. While 332
HA-tagged Usp15 co-precipitated Flag-tagged WT Hrd1, the ΔTM mutant lost 333
interaction with Usp15 (Figure 4e). With these different strategies, we have demonstrated 334
that Hrd1 directly interacts with Usp15 depending on Hrd1’s transmembrane domain, and 335
that this interaction is enhanced by TLR4 stimuli. 336
Because the crystal structure of Usp15 (PDB 4A3O)38 and the cryo-EM structure of 337
Hrd1 (PDB 5V6P)35 have been demonstrated, we further propose a model to describe the 338
interaction between Usp15 and Hrd1: a concave surface is formed by the transmembrane 339
helices of Hrd1, the DUSP and UBL domains of Usp15 might bind this concave surface 340
and lie on each side of the Hrd1 dimer (Figure S4i). 341
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Because Hrd1 regulated TLR4-triggered IκBα degradation in macrophages, we next 342
investigated whether its binding partner Usp15 was also involved in the TLR4 pathway. 343
Knock-down of Usp15 promoted LPS-induced production of IL-6, TNF-α and IL-1β at 344
the mRNA level (Figure 4f) as well as LPS-induced IκBα degradation (Figure 4g, left 345
panel), without affecting TLR4 expression in PEMs (Figure 4g, right panel). Importantly, 346
Usp15 knock-down rescued the reduced IL-6 production in Hrd1-deficient macrophages 347
upon LPS stimulation (Figure 4h). Hrd1-deficient macrophages did not affect Usp15 348
mRNA levels (Figure S4j; the knock-down efficiency of Usp15 was shown). To further 349
ask whether Usp15 regulated TLR4-induced inflammation mainly via NF-κB, we next 350
knocked down the NF-κB subunit p65 using two different siRNAs in Usp15 KD 351
macrophages and observed that knock-down of p65 substantially blocked the enhanced 352
IL-6 production in Usp15 KD macrophages by ELISA (Figure 4i) or by RT-PCR (Figure 353
S4k) assays. The knock-down efficiencies of Usp15 or p65 were shown in Figure S4k 354
(lower panels). Furthermore, the NF-κB luciferase activity was gradually reduced when 355
an increasing amount of Usp15 was co-expressed (in a dose-dependent manner) with 356
Hrd1 in HEK293T cells in the presence of IKKβ (Figure S4l, Usp15 expression levels 357
were shown in the lower panel). 358
Usp15 is a DUB, and previous studies have suggested that the C298A/C812A 359
catalytically inactive Usp15 mutant (termed M2 in this study) loses its deubiquitination 360
ability39. We next asked whether Usp15 DUB activity is crucial for Hrd1-mediated 361
NF-κB activation. Overexpression of Usp15 reduced Hrd1-induced NF-κB luciferase 362
activity; in contrast, overexpression of the Usp15 M2 mutant failed to inhibit 363
Hrd1-mediated effects in the presence of IKKβ (Figure 4j). These data suggest that Hrd1 364
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directly interacts with Usp15 in macrophages and that the DUB activity of Usp15 is 365
critical for Hrd1-induced IκBα degradation and proinflammatory cytokine production, 366
specifically via the TLR4 pathway. 367
368
Hrd1 promotes polyubiquitination of Lys21 in Usp15 and inactivates Usp15 369
Usp15 is a DUB and, interestingly, its DUB activity is critical for Hrd1 to modulate 370
inflammation (Figures 4j). We next asked whether Hrd1 ubiquitinates Usp15, thus 371
affecting its DUB activity or degradation. To elucidate this, we first co-expressed WT 372
Hrd1, the C329S or ΔTM mutant with HA-tagged Usp15 and His-tagged ubiquitin (Ub) 373
in HEK293T cells, followed by immunoprecipitation with anti-HA antibody. When 374
overexpressed with WT Hrd1 in HEK293T cells, HA-Usp15 ubiquitination levels were 375
significantly enhanced compared to the GFP control, the Hrd1 catalytic inactive mutant 376
C329S and ΔTM mutant (Figure 5a, left panel). To confirm that the signals came from 377
ubiquitinated Usp15, we incubated the immunoprecipitated HA-Usp15 with the catalytic 378
core of human Usp2 (Ubiquitin-specific protease 2) (termed Usp2cc). Hrd1-induced 379
ubiquitination of Usp15 was indeed eliminated after incubation with Usp2cc (Figure 5a, 380
middle panel). His-tagged Usp15 was purified with Ni-NTA beads with the buffer 381
containing high concentrations of salt to minimalize the possibility of pulling down its 382
binding partners, followed by the Usp15 ubiquitination assays to confirm that the 383
detected ubiquitin conjugates were bound to Usp15 itself but not Usp15-associated 384
proteins (Figure 5a, right panel). 385
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To further confirm that the DUB Usp15 is a substrate of Hrd1, we constituted the E1 386
Ub-activating enzyme (Uba1), the E2 Ub-conjugating enzyme (Ube2g2), the E3 WT 387
Hrd1 or the C329S mutant, HA-tagged Ubiquitin and His-tagged catalytically inactive 388
Usp15 (i.e., C298A/C812A mutant, termed Usp15 M2) into E. coli40, 41. Using the 389
assembled ubiquitin system in E. coli, His-tagged Usp15 was purified with Ni-NTA 390
beads using the buffer containing high concentrations of salt, followed by the in vitro 391
Usp15 ubiquitination assays. We indeed observed that WT Hrd1, but not the C329S 392
mutant, successfully ubiquitinated Usp15 (Figure 5b). Furthermore, the E3 ligase HACE 393
was used as a negative control and constituted into E. coli. HACE could catalyze free 394
ubiquitin conjugation to ubiquitin chains (Figure S5a) but could not catalyze Usp15 395
ubiquitination, which showed no ubiquitination signals in Usp15 (Figure 5b, last lane). 396
Together, we have confirmed that Hrd1 induces Usp15 ubiquitination by using the 397
eukaryotic HEK293T cells and a reconstituted prokaryotic system. Next, we 398
immunoprecipitated Usp15 in PEMs and immunoblotted with anti-ubiquitin antibody. 399
LPS stimulation significantly increased Usp15 ubiquitination levels (Figure 5c). 400
Importantly, in comparison to WT PEMs, the Usp15 ubiquitination signal was strongly 401
reduced in Hrd1-deficient macrophages (Figure 5d). This result was consistent with 402
enhanced Usp15 translocation to the ER (Figures 4d and S4e) and increased Usp15 403
interaction with Hrd1 (Figures 4c and S4b) after LPS treatment. Together, these results 404
demonstrate that Hrd1 binds Usp15 to promote Usp15 ubiquitination in macrophages.The 405
next important question was which lysine residues on Usp15 are ubiquitinated by Hrd1. 406
Using the E. coli reconstituted ubiquitin system, Hrd1-induced ubiquitinated Usp15 was 407
enriched by two rounds of immunoprecipitation using Ni-NTA (Usp15) and anti-HA 408
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(Ubiquitin) antibodies then subjected to trypsin digestion followed by MS analysis. The 409
MS results identified ubiquitination sites at Lys21, Lys154 and Lys228 in Usp15. For 410
verification of these results, we generated Usp15 mutants bearing the individual 411
Lys-to-Arg substitutions, including K21R, K154R and K228R. We first found that these 412
substitution mutants of Usp15 formed stable complexes with Hrd1 via 413
immunoprecipitation assay in HEK293T cells (Figure S5b). By contrast, only when 414
Lys21 was mutated to Arg (i.e., K21R), Usp15 ubiquitination was eliminated (Figure 5e). 415
We next investigated the biological consequences of Hrd1-mediated ubiquitination 416
of Lys21 in Usp15. Unexpectedly, unlike the Hrd1 targets for ERAD, Hrd1-induced 417
ubiquitination of Usp15 did not lead to Usp15 degradation (Figure S5c). The Lys21 418
residue is located in the DUSP domain of Usp15, and previous studies have reported that 419
Usp15 requires the DUSP-Ubl domain to achieve its full catalytic efficiency42. This 420
raised the key question of whether Lys21 polyubiquitination regulated Usp15 catalytic 421
activity. We co-expressed Myc-tagged Hrd1 or the C329S mutant with Flag-tagged 422
Usp15 or the K21R mutant in HEK293T cells then purified Flag-Usp15 by 423
immunoprecipitation. During the immunoprecipitation process, we added excess 424
polyubiquitin chains which competitively protected Usp15 polyubiquitination (Figure 425
S5d). Using DUb-7-amido-4-methylcoumarin (DUb-AMC) as the substrate, we found 426
that activated Usp15 cleaved DUb-AMC and released a C-terminal derivatization of 427
ubiquitin with 7-amino-4-methylcoumarin. By measuring fluorescence intensity, we 428
observed that ubiquitinated Usp15 had significantly reduced DUB activity levels 429
compared to the K21R mutant or the un-ubiquitinated Usp15 (Figure 5f). We next used 430
another strategy to examine how Usp15 ubiquitinatation affected its DUB activity. Indeed, 431
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when a K48-linked polyubiquitin chain (Ub2-16) was used as a substrate, the K21R mutant 432
showed higher activity in deubiquitinating polyUb (i.e., Ub4, 5, or Ub 6-Ub16) with an 433
enhanced amount of mono-Ub (Figure 5g, samples with long exposure). Next, NF-κB 434
luciferase reporter assays were performed to determine the biological function of Lys21 435
ubiquitination in Usp15. Compared to WT Usp15, the non-ubiquitinated K21R mutant 436
displayed a better inhibitory effect on Hrd1-induced luciferase activity in a 437
dose-dependent manner (Figure5h, increasing expression levels of Usp15 and K21R were 438
shown in the lower panel). Indeed, when comparable levels of WT Usp15 or the K21R 439
mutant were stably overexpressed in MEFs (Figure5i, right lower panel), the K21R 440
mutant showed a further decrease of Il6 production at the mRNA and protein levels after 441
E. coli treatment (Figure 5i, left two panels). Compare to the GFP control sample, stably 442
overexpression of WT Usp15 could remove ubiquitin in IκBα, resulting in stabilization 443
IκBα in response to LPS treatment (Figure S5e). The K21R mutant could further stabilize 444
IκBα, comparing to that in WT Usp15, but the M2 mutant lost the DUB activity and 445
maintained LPS-induced IκBα degradation (Figure S5e). These results agree with the 446
enhanced DUB activity of the K21R mutant (Figures 5f and 5g). Together, we have 447
demonstrated that Hrd1 inhibits Usp15 DUB activity by adding Lys27-linked 448
polyubiquitin chains to the Lys21 residue in Usp15. 449
450
Hrd1 deficiency protects against LPS- and CLP- induced septic shock 451
Since excessive TLR4-NF-κB activation plays a critical role in sepsis, we asked 452
whether Hrd1 deficiency in macrophages protects against LPS- and CLP-induced septic 453
shock in mice. Upon intra-peritoneal injection of LPS, more Hrd1 cKO mice survived 454
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compared to the WT littermate controls (Figure 6a). In agreement with the notion that an 455
uncontrolled inflammatory response is critical for induction of septic shock, we observed 456
significantly lower concentrations of serum IL-6 in Hrd1 cKO mice (Figure 6b). 457
Similarly, Hrd1 cKO mice exhibited less robust Il6 production in multiple organs, 458
including the liver, lung and kidney (Figure 6c). Immunohistological studies showed that 459
the WT mice displayed more severe lung injury than Hrd1 cKO mice (Figure 6d). 460
To confirm the role of Hrd1 in pathogen-induced septic shock, the WT and cKO 461
littermates were subjected to caecal ligation and puncture (CLP) surgery. In agreement 462
with the results from LPS-induced septic shock, Hrd1 cKO mice died at later time points 463
and had increased survival rates (Figure 6e). IL-6 concentrations in serum (Figure 6f) as 464
well as mRNA levels in the liver, lung, kidney and spleen (Figure 6g) were significantly 465
reduced in Hrd1 cKO mice compared to their WT littermates 6 hr after CLP operation. 466
Immunohistological staining of the lungs showed enhanced lung damage in WT mice 24 467
hr post CLP surgery (Figure 6h). 468
We next asked a critical question: how Usp15 deficiency affects Hrd1 KO 469
macrophage function in vivo. Because Usp15 and Hrd1 double KO mice were not 470
available, we depleted endogenous peritoneal macrophages via intra-peritoneal (i.p.) 471
injection of clodronate-containing liposomes followed by adoptive transferring of 472
CFSE-labeled WT or Hrd1-deficient macrophages transfected with scrambled siRNA or 473
siUsp15. We confirmed that treatment with clodronate-containing liposomes resulted in 474
marked depletion of endogenous F4/80+CD11b+ macrophages in the peritoneal cavity 475
(Figure S6a). Usp15 KD efficiency was confirmed in WT and Hrd1 KO PEMs (Figure 6i, 476
right panel). After adoptively transferring Hrd1 KO/Usp15 KD PEMs for 24 hr, mice 477
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were challenged with LPS for 6 hr followed by collection of CFSE+ PEMs. In agreement 478
with the in vitro data, Hrd1 KO macrophages exhibited lower IL-6 production (Figure 6i, 479
left panel). Importantly, knock-down of Usp15 rescued reduced Il6 production in Hrd1 480
KO macrophages upon LPS treatment, which reached levels similar to those in WT cells 481
(Figure 6i, left panel). 482
We previously excluded the role of Hrd1 in TLR3/TLR9-induced inflammation in 483
vitro (Figure 2h). To further examine whether Hrd1 regulated TLR3/9 signalling in vivo, 484
we injected CpG or Poly(I:C) respectively, into WT and Hrd1 cKO mice through the tail 485
vein. Consistent with the in vitro data, serum IL-6 or IFN-β concentrations were not 486
significantly changed between the paired WT and Hrd1 cKO mice (Figures 6j and 6k, left 487
panel), and mRNA levels of Il6 and Ifnb in multiple organs were also comparable 488
(Figures 6j and 6k, right panels). These results confirmed that the Hrd1/Usp15 module 489
also specifically affected TLR4 signalling in vivo. 490
Together, our data have elucidated a model linking the TLR4 innate signalling 491
from the cell membrane to the ER (Model in Figure 6l). As an ER-located E3 ligase, 492
Hrd1 is important for transducing signals from the cell membrane receptor TLR4 to the 493
DUB Usp15, which is recruited to the proximal ER membrane to induce IκBα 494
degradation and NF-κB activation. 495
496
Discussion 497
More and more E3 ubiquitin ligases are being identified to precisely regulate 498
TLR4-NF-κB activation, such as TRAF6 and Pellino-1. In this study, we have identified 499
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that Hrd1, the ER-located RING-type E3 ligase, is critical for the plasma membrane 500
receptor TLR4-induced signalling and proinflammatory cytokine production in 501
macrophages. Deletion of the Hrd1 gene or knock-down of Hrd1 expression specifically 502
attenuated IL-6, TNF-α and IL-1β production in macrophages after LPS treatment, 503
Salmonella typhimurium or E. coli infection. Interestingly, Hrd1 deficiency did not 504
modulate IL-6 or IFN-β production after activation of the TLR2/TLR3/9 pathways. In 505
vivo Hrd1-specific deficiency in myeloid cells protected mice from LPS- or CLP-induced 506
septic shock, suggesting that targeting Hrd1 might be applied to protect the host from 507
severe bacterial infections. 508
Expression of Hrd1, also known as Syvn1, is increased in synoviocytes and 509
peripheral blood cells from RA patients19, and this increase is correlated with the onset of 510
RA. Previous work suggests that Hrd1+/− mice increase apoptosis of synovial cells to 511
protect against collagen-induced arthritis20, 21. In contrast to Hrd1 KO synovial cells, we 512
did not observe abnormal apoptosis in Hrd1 KO macrophages. Importantly, our study 513
suggests that Hrd1 acts as a positive regulator for IL-6 and TNF-α production via the 514
TLR4 pathway in macrophages. Previous immunohistological analyses of inflamed RA 515
joint tissue have demonstrated that TLR4 is highly expressed in infiltrated macrophages22, 516
43, and macrophages are one of the main sources of proinflammatory cytokines44. IL-6 517
and TNF-α play crucial roles in the pathology of RA, and the most widely used biologic 518
anti-rheumatic drugs are anti-TNF agents. It will be intriguing to examine whether Hrd1 519
affects IL-6 and TNF-α production in infiltrated macrophages from inflamed RA joints. 520
We therefore propose that inhibition of Hrd1 E3 ligase activity might be a promising 521
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approach to treat RA by targeting two major cell types, including increasing apoptosis of 522
synovial cells and proinflammatory cytokine secretion in macrophages. 523
Our study has identified Hrd1 as an ER-located protein that regulates 524
TLR4-signalling during bacterial infection in vivo and in vitro. Cellular localisation is 525
important for certain proteins to perform their functions, including the 526
mitochondria-located MAVS4, the ER-located STING6, 7 and the endosome-located 527
TLR3 that participate in the innate immune response against viruses. In particular, several 528
key amino acids substitutions in STING were mapped from patients exhibiting 529
autoimmunity. Interestingly, these residues make up a small linker region connecting the 530
N-terminal transmembrane domain of STING to the C-terminal cyclic 531
dinucleotide-binding domain45. These residues are critical in retaining STING on the ER 532
in unstimulated cells, and disease mutations disrupt ER retention46 and causing abnormal 533
interferon response6, 47. The ER has a broad localisation throughout the cell and can form 534
direct physical contacts with all other membranous organelles2, 3, 6, 10, 11, which are 535
involved in lipid exchange between membranes, controlling Ca2+ homeostasis and other 536
key biological functions. In the past several years, advancing studies focus on the 537
structure and function of membrane contact sites within cells, including the contact of the 538
ER with mitochondria and endosome10, 11. However, it is largely unknown whether ER 539
functions during bacterial infection and which molecules with special ER localisation 540
could modulate the TLR4 pathway or anti-bacterial infection. Our study provides an 541
interesting example on how two membrane proteins (i.e. TLR4 and Hrd1) from the 542
contacts of the ER with the cell plasma membrane function in regulating bacterial 543
infection and inflammation. This provides an important hint linking ER with 544
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anti-bacterial infection. Hopefully it will trigger more interest in understanding the 545
biology and function of the endomembrane system as well as the crosstalk between ER 546
and the plasma membrane. 547
As an ER-located E3 ligase, the classical role of Hrd1 is to ubiquitinate incorrectly 548
folded proteins in the ER to promote their degradation and to prevent ER-stress-induced 549
cell apoptosis. Our study has illuminated that unlike the role of Hrd1 in ERAD, Hrd1 in 550
LPS-stimulated macrophages catalyses Usp15 ubiquitination at Lys27 which does not 551
lead to Usp15 instability and degradation, but rather inactivates Usp15. This might open a 552
research angle for studying the ERAD-independent function of Hrd1. 553
Prior studies have implicated Usp15 in the regulation of a variety of cellular 554
signalling pathways, including the TGF-β, NF-κB, β-catenin, spliceosome and p53 555
signalling pathways39, 48. Usp15 dysregulation has been demonstrated in cancer and 556
immune responses26, 49. Despite its critical functions, the mechanism of Usp15 regulation 557
has not yet been fully elucidated. In this work, we demonstrated that Usp15 translocates 558
to the ER and is modified by Hrd1-mediated polyubiquitination after TLR4 activation in 559
macrophages. The ER-located E3 Hrd1 catalyses the Lys21 in Usp15, disrupting its DUB 560
activity and preventing it from deubiquitinating downstream effectors, including IκBα. 561
The Lys21 in Usp15 represents the first-identified post-translational modification of 562
Usp15. In addition, recent studies have implicated ER dysfunctions and ER stress are 563
involved in liver diseases, Alzheimer’s disease, cancer32, 50. This suggests that inhibit ER 564
dysfunctions or targeting the Hrd1/Usp15 module might be useful in treating related 565
pathological conditions. 566
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In summary, we have provided compelling evidence that the ER-located E3 567
ubiquitin ligase Hrd1 ubiquitinates the DUB Usp15 to modulate TLR4-NF-κB signalling 568
in macrophages in response to Gram-negative bacterial infection. Given the role of Hrd1, 569
Usp15 and the proinflammatory cytokines IL-6 and TNF-α in pathological conditions 570
including rheumatoid arthritis, Parkinson’s disease, cancer and anti-pathogen infections, 571
this study might provide therapeutic hints for drug design. 572
573
Material and methods 574
Mice. The sixth exon of Hrd1 gene was flanked by two loxP sites to generate Hrd1 575
conditional knock-out mice (the F0 generation i.e. Hrd1fl/- mice) (Sidansai Biotechnology 576
Company, Nanjing University). Hrd1fl/- mice were crossbred with LyzM-Cre+/+ mice to 577
generate Hrd1fl/-LyzM-Cre+/- mice, which were then backcrossed with Hrd1fl/- mice. The 578
Hrd1fl/flLyzM-Cre+/- cKO mice specifically knocked out Hrd1 in myeloid cells, and the 579
littermate Hrd1fl/flLyzM-Cre-/- mice were used as the controls. Genotyping was performed 580
with the following primers: forward: 5′-GCATAGTCTCTACTCTGTTC-3′; reverse: 581
5′-CTTCCTGCTCCACGACAATC-3′. The mice were on C57BL/6 background. All 582
mice were maintained under specific pathogen-free conditions with approval of the 583
institutional animal facility of Shanghai Institute of Biochemistry and Cell Biology 584
(protocol IBCB0057) and the National Institute for Viral Disease Control and Prevention. 585
Animals were randomly allocated to experimental groups. The animal experiments 586
performed with a blinded manner were described below. Statistical methods and advice 587
from the related publications were considered to determine the number of mice used. All 588
animal experiments were approved by the Institutional Animal Care and Use Committee 589
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27
(IACUC) of Institute of Biochemistry and Cell Biology, Shanghai Institutes for 590
Biological Sciences, Chinese Academy of Sciences. 591
Cells and reagents. Cells were maintained at 37 °C under 5% CO2. HEK293T cells were 592
kindly provided by Dr. Xiaohui Zhang (SIBCB, CAS). Primary MEF cells as well as 593
MEF cell lines were kind gifts from Dr. Anning Lin (SIBCB, CAS). Primary peritoneal 594
macrophages, HEK293T, primary MEF, MEF cell lines were tested and confirmed no 595
mycoplasma contamination, which were cultured in Dulbecco’s modified Eagle 596
medium (DMEM) supplemented with 10% (v/v) FBS, L-Glutamine (2m M), and 597
penicillin–streptomycin (100 U/ml). THP-1 cells were cultured in complete RPMI 1640 598
medium, induced by Phorbol-12-myristate-13-acetate (PMA) (final concentration 300 599
ng/ml) for 12 hr to differentiate to macrophages. Mouse peritoneal macrophages were 600
prepared from 12 weeks old mice through intraperitoneal injection with 3 ml 3% Brewer 601
thioglycollate medium. To generate bone-marrow-derived macrophages (BMM), bone 602
marrow cells were cultured with 30% L929-conditioned media for a week. All cell lines 603
used in this study were not contaminated by mycoplasma, which were examined by 604
PCR-based detection of mycoplasma. Lipopolysaccharide was from Escherichia coli 605
055:B5 and purified by phenol extraction. CpG-B (ODN1826) and Poly(I:C) (HMW) 606
Rhodamine and PGN (tlrl-pgnb3, InvivoGen) were from Invivogen. Clodronate 607
liposomes were from FormuMa. Antibodies used in this study were listed in Table S2. 608
Plasmids. cDNAs for human HRD1, USP15, NFKBIA were amplified from 609
reverse-transcribed cDNA from HEK293T cells. These genes were cloned into the 610
pcDNA3.1-IRES-GFP vector for transient expression in HEK293T cells. For stable 611
expression, human HRD1 and USP15 were subcloned into the pMSCV-IRES-GFP vector 612
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28
with Flag tag and HA tag respectively. For recombinant expression in E.coli, USP15 was 613
inserted into the pET-Duet vector with 6xHis tag. Mutations in HRD1 or in USP15 were 614
constructed by standard PCR cloning strategy. Plasmids expressing ubiquitin 615
lysine-to-arginine mutations were kindly provided by Dr. Ronggui Hu (SIBCB, CAS). 616
Usp15 truncation mutations were kindly gifts from Dr. Shaocong Sun (MD Anderson 617
Cancer Center, USA). All plasmids were confirmed by DNA sequencing. Plasmids used 618
in this study were listed in Table S3. 619
RNAi. siRNA (Dharmacon) targeting specific genes were delivered into PEMs and 620
BMMs by Lipofectamine RNAiMAX Reagent (Invitrogen) according to the 621
manufacturer’s instructions. Sequences of all siRNA were listed in Table S4. 622
Quantitative RT-PCR analysis. Total RNA was isolated by RNAiso Plus regent 623
according to the manufacturer’s instructions (TAKARA). First-strand cDNA was 624
generated using M-MLV reverse transcriptase (RNase H-) (TAKARA). Samples were 625
amplified by CFX-96 machine (Bio-rad) using SYBR Green master mix (DBI Bioscience) 626
according to the manufacturer’s instructions, and data were normalized to an Actb control. 627
Sequences of all primers were listed in Table S5. 628
Stable cell line establishment and BMM transduction. Retroviral particles were 629
prepared by transfecting HEK293T cells with pCL-10A and MIGR or MSCV vectors 630
expressing target genes. MEFs were infected with retroviral supernatants in the presence 631
of polybrene (final concentration 1 μg/ml) (Santa Cruz). Cells overexpressing the 632
indicated genes were selected by sorting GFP positive cells or by puromycin (final 633
concentration 1.5 μg/ml) treatment. To generate stably transduced BMMs, 634
L929-conditioned media was supplemented with retroviral supernatants at 1:1 ratio to 635
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29
culture BM cells at the first 3 days. After 7 days induction, BM cells were derived into 636
macrophages which stably overexpressed the indicated genes. 637
ELISA. Concentrations of mouse IL-6 or TNF-α in serum or cell supernatants were 638
measure by ELISA commercial kits (eBioScience) according to the manufacturer’s 639
instructions. 640
Dual-luciferase reporter assay. HEK293T cells were transfected with the NF-κB 641
luciferase reporter, TK-renilla and the indicated plasmids by Polyethylenimine 642
(Polysciences) as the manufacturer’s instructions. Cells were harvested after 24 hr to 643
measure luciferase readings with a dual-luciferase reporter assay system according to the 644
manufacturer’s instructions (Promega). 645
Isolation of cytoplasmic and nuclear fractions. PEMs or PMA-induced THP-1 cells 646
were stimulated with 1 μg/ml LPS for the indicated time points. Cells were harvested, 647
washed with PBS, then lysed with buffer A (10 mM HEPES, 1.5 mM MgCl2, 10 mM 648
KCl, 0.5 mM DTT, 1 mM PMSF, 0.1% (v/v) NP-40 and protease inhibitor cocktail, pH 649
7.9). Lysates were centrifuged at 6,000 rpm for 15 min at 4°C. Supernatants containing 650
cytoplasmic proteins were collected. Nuclear proteins were extracted from the pellet by 651
cold buffer C (20 mM HEPES, 1.5 mM MgCl2 , 0.42 M NaCl, 0.2 mM EDTA, 25% (v/v) 652
glycerol, 0.5 mM DTT, 1 mM PMSF and protease inhibitor cocktail, pH 7.9), and 653
insoluble material was removed by centrifugation at 12,000 rpm for 1 min at 4°C. 654
Immunostaining. MEF cells or PEMs were stimulated with 1 μg/ml LPS as the indicated 655
time points, mounted on coverslips, fixed, permeabilized, and then stained with the 656
indicated antibodies and Hoechst. Images were taken by Olympus IX81 microscopy or 657
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Leica confocal microscopy. Fluorescent imaging analysis was conducted in a blinded 658
fashion, and densitometry quantification or Pearson’s correlation was analyzed by 659
Image-Pro 6.0 software. 660
Mass spectrometry analysis. To identify the substrates of Hrd1, THP-1 cells which 661
stably overexpressed Flag-dsRed or Flag-Hrd1 were treated with PMA (final 662
concentration 300 ng/ml) for 12 hr to derive into macrophages. After washing, adherent 663
THP-1 macrophages were stimulated with 1 μg/ml LPS for 1hr and then lysed with lysis 664
buffer (20 mM Tris-Cl, 100 mM KCl, 1 mM EDTA, 0.1% (v/v) NP-40, 10% (v/v) 665
glycerol, and protease inhibitor cocktail, pH 7.5). Whole cell extracts were 666
immunoprecipitated with anti-Flag beads. Proteins were eluted by the Flag peptides and 667
subjected to make freeze-dried powder. 668
To investigated which lysines in Usp15 were ubiquitinated by Hrd1, BL21 669
competent cells transferred with indicated plasmids were inoculated and induced by 670
IPTG (1 mM) at 16 °C for 16 hr. Bacteria harvested in RIPA buffer were lysed by sonic 671
and centrifuged at 4 °C for 15 min. Following incubation of cell lysates and Ni-NTA 672
beads at 4 °C for 2 hr, the beads were washed for five times with RIPA buffer and eluted 673
with 200 mM imidazole. The eluent was dialyzed in PBS containing 10% glycerol at 674
4 °C overnight and collected the liquid in dialysis bag. Next, the resulting proteins were 675
immunoprecipitated with anti-HA beads to enrich ubiquinatinated Usp15. The beads 676
were washed for five times with RIPA buffer, and a part of the beads were harvested with 677
SDS loading buffer and the sample were determined by western blot. Protein pellet was 678
dissolved in 10 μl of 8 M Urea, 100 mM Tris-HCl, pH 8.5, and diluted to 80 μl 1M Urea 679
with 100 mM Tris-HCl, pH 8.5. The pH was adjusted to pH 1.0 by adding 1 M HCl. 680
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31
Digestion was performed in the presence of 100 mM Tris-HCl using sequencing-grade 681
soluble trypsin (Roche). 682
Yeast two-hybrid. To examine the interaction between Hrd1 and Usp15, GAL4-based 683
yeast two-hybrid system (ProQuest™ Two-Hybrid System, Invitrogen) were applied. 684
Full length human Hrd1 ORF (open reading frame) was cloned into the donor vector 685
pDONR221 and transfected into pDEST32 via Gateway cloning reaction (Invitrogen). In 686
pDEST32 plasmid, Hrd1 was in-frame fused with GAL4 DNA binding domain, 687
generating the bait plasmid. Human Usp15 was in-frame fused to the GAL4 activation 688
domain (Invitrogen) in prey vector pDEST22. To perform the Yeast two-hybrid assay, 689
the yeast strain Mav203 competent cells were transformed with the bait clone 690
pDEST32-Hrd1 and the prey clone pDEST22-Usp15. The empty vectors pDEST22 and 691
pDEST32 were used as the negative prey or bait controls. pEXP32-Krev1 and 692
pEXP22-RalGDS-WT were used as the positive controls. Mav203 competent cells that 693
were transformed with the bait and prey plasmids could grow and form clones on 694
SC-Leu-Trp (SD-2) plates, while only cells containing the interacting proteins could 695
grow on SC-Leu-Trp-His-Ura (SD-4) plates. When pDEST32- Hrd1 and 696
pDEST22-Usp15 were transformed into Mav203 competent cells, all reporter genes were 697
activated. 698
Expression and purification of recombinant proteins. The full length and the 699
transmembrane domain truncation mutant (ΔTM) of human Hrd1 were constructed to the 700
pGEX4T-1-GST vector (GE Healthcare) to generate GST-fusion proteins. Human Usp15 701
was cloned into pET-Duet-His vector. Recombinant proteins were expressed in E. coli 702
BL21 (DE3) Codon-Plus strain (Novagen). BL21 cells were transformed with the above 703
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32
plasmids and grown in L-broth supplemented with ampicillin (50 μg/ml). Expression of 704
the recombinant proteins was induced with 0.1 mM IPTG at 16 °C for 20 hr. For 705
purification, 6×His-Usp15 was purified by Ni-NTA affinity chromatography (Qiagen, 706
Germany); GST-Hrd1, GST-ΔTM or GST were purified by Glutathione-agarose beads 707
according to the manufacturer’s instructions (GE). Purified Hrd1 protein was identified 708
by western blotting without boiling. 709
GST pull-down. GST-Hrd1 (3 μg), GST-Hrd1-ΔTM (2.4 μg) or GST proteins (0.92 μg) 710
at equimolar concentrations were incubated with His-Usp15 (4 μg) at 4 °C for 2 hr in 100 711
μl pull-down buffer (20 mM Tris-Cl, 100 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM 712
DTT, 0.5% (v/v) NP-40 and 10 μg/mL BSA, pH 7.5) followed by washing three times. 713
Samples were combined with SDS loading buffer and subjected to SDS-PAGE without 714
boiling. 715
Generation of the interaction model. The interaction model between Usp15 and Hrd1 716
complex was generated with the Pymol program (The PyMOL Molecular Graphics 717
System, Version 1.6, Schrödinger, LLC). 718
Immunoprecipitation analysis. HEK293T cells overexpressing the indicated proteins 719
were washed with cold PBS before lysed with cold lysis buffer (25 mM Tris-Cl, 150 mM 720
NaCl, 1% (v/v) NP-40, 5 mM EDTA, 0.5% sodium deoxycholate and protease inhibitor 721
cocktail, pH 7.2). Cell lysates were then centrifuged at 13, 500 rpm for 15 min, 4 °C. 722
Following incubation of cell lysates with protein G Sepharose coated with indicated 723
antibodies and rotating at 4 °C for 2 hr, beads were then washed for five times with lysis 724
buffer and resuspended in SDS-PAGE loading buffer for western blot analysis. 725
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Cellular fractionation. PEMs were harvested into 5 ml ice-cold PBS and centrifuged at 726
1,000 g for 5 min at 4 °C. Cell pellets were resuspended in 0.4 ml Buffer F (10 mM 727
HEPES-KOH at pH 7.2, 250 mM sorbitol, 10 mM KOAc, 1.5 mM Mg(OAc)2 and 728
protease inhibitor cocktail), passed through a 22-gauge needle 20 times, incubated on ice 729
for 15 min, and centrifuged at 1,000 g for 5 min at 4 °C. Supernatants were transferred to 730
microtubes, and centrifuged at 16,000 g for 3 min at 4°C. The pellets were resuspended 731
in 0.5 ml Buffer E (50 mM HEPES-KOH at pH 7.2, 250 mM sorbitol, 70 mM KOAc, 5 732
mM potassium EGTA, 2.5 mM Mg(OAc)2 and protease inhibitor cocktail), and 733
centrifuged at 16000 g for 3 min at 4 °C to obtain microsomes. 734
EM Data Collection. LPS (1 μg/ml) was used to coat latex beads (3 μm, Sigma) via 735
shaking at 4 °C overnight. PEMs were treated with LPS-coated or non-coated latex beads 736
for 60 min, and fixed with 2.5% glutaraldehyde in PBS for 1.5 hr at 4 °C. After washing 737
with PBS three times, the fixed samples were post-fixed with 1% osmium tetroxide for 1 738
hr at 4 °C. The samples were washed with PBS three times and dehydrated in an ethanol 739
series (25 %, 50 %, 75 %, 95 % ethanol in distilled water, each for 10 min). Then the 740
samples were dehydrated in pure ethanol twice (each for 30 min) and infiltrated with 741
ethanol:epoxy 812 (at 2:1, 1:1 and 1:2 ratio, each for 30 min), and then infiltrated with 742
pure epoxy 812 overnight. Then the samples were infiltrated with fresh epoxy 812 for 1 743
hr and embedded in epoxy 812. After polymerizing at 65 °C for 48 hr, the resin blocks 744
were trimmed and thin sectioned with a thickness of 70 nm on an ultramicrotome using a 745
diamond knife. The thin sections were mounted onto formvar-coated copper grids and 746
counterstained with 3 % uranyl acetate in 70 % methanol for 7 min and then by lead 747
Page 34
34
citrate for 3 min. The stained sections were viewed under a FEI electron microscope 748
(Tecnai G2 Spirit 120kV) and images were recorded on a FEI Eagle CCD camera. 749
In vivo ubiqunitination assay. For in vivo ubiquitination of Usp15, HEK293T cells 750
transiently transfected with HA-tagged Usp15, His-tagged WT or mutant ubiquitin and 751
Flag-tagged Hrd1 or its E3 ligase-dead mutant C329S or PEMs lysed in RIPA buffer (50 752
mM Tris-Cl, 150 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X-100, 0.5% sodium 753
pyrophosphate, 0.1% SDS, and protease inhibitor cocktail, pH 7.4) containing 10 mM 754
N-Ethylmaleimide. Cell lysates were immunoprecipitated with anti-HA beads or with 755
protein G Sepharose coated with 2 μg anti-Usp15 antibody/sample and then washed for 756
five times with RIPA buffer, followed by immunoblotting analysis to detect indicated 757
proteins. For Figure 5a left panel, after the immunoprecipitation, HA beads were 758
resuspended in PBS buffer and subjected to Usp2cc (catalytic core of Usp2) digestion 759
(final concentration 100 μg/ml), followed by immunoblot analysis. For Figure 5a right 760
panel and Figure 5b, HEK293T cells and E.coli overexpressing His-Usp15 and the 761
indicated proteins were suspended in denaturing lysis buffer (6 M Guanidine 762
Hydrochloride, 20 mM Sodium Phosphate, pH 7.8, 500 mM NaCl) and lysed by 763
sonication. Cell lysates were incubated with Ni-NTA beads at 4 °C for 2 hr, and washed 764
three times with binding buffer ( 8 M Urea, 20 mM Sodium Phosphate, pH 7.8, 500 mM 765
NaCl) and wash buffer (8 M Urea, 20 mM Sodium Phosphate, pH 6.0, 500 mM NaC), 766
respectively. 767
LPS shock and CLP model. To monitor survival conditions, 8 weeks old male WT and 768
cKO mice were injected intraperitoneally with LPS (35 mg/kg) to induce LPS shock. To 769
generate the CLP model, mice were anesthetized, and an abdominal incision was made 770
Page 35
35
for identification of the cecum. The distal one third of the cecum was ligated with silk 771
suture and was punctured twice with a 21-gauge needle. A small amount of cecal content 772
was extruded through the perforation. The peritoneum and skin were closed with 773
autoclips (Becton Dickinson) after the cecum was returned to the abdomen. 1 ml saline 774
was injected intraperitoneally for resuscitation. For sham-treated mice, all of the same 775
steps were performed, except for ligation and puncture of the cecum. 776
Adoptive macrophage transfer. 8-week-old WT male mice were injected 777
intraperitoneally with 100 μl of Clodronate liposomes (FormuMax) to delete peritoneal 778
macrophages and used as the recipient. PEMs obtained from WT and cKO mice were 779
transfected with scramble or mouse siUsp15 in a 1.5 ml Eppendorf tube for 6 hr followed 780
by CFSE labeling. Equal numbers of the control or siUsp15-transfected CFSE+ PEMs (10 781
million) were i.p injected to the Clodronate liposome-treated recipient mice. 24 hr later, 782
mice were i.p challenged with 25 mg/ kg LPS for 6 hr, sacrificed and adoptively 783
transferred CFSE+ PEMs were harvested to extract mRNA for RT-PCR analysis. 784
In vivo Poly(I:C) and CpG treatment. Age- and sex-matched adult mice were i.v 785
injected with 100 μg Poly(I:C)/ mouse or 100 μg CpG-B/mouse. Mice were sacrificed on 786
the indicated time points, serum and organs were harvested to measure expression levels 787
of IL-6 and IFN-β. 788
Flow cytometry. All samples passed through a 70 µm nylon cell strainer to make 789
single-cell suspension. For surface staining, cells were labeled with the indicated 790
antibodies in FACS buffer (2.5 L PBS, 50 ml NBS and 0.5 g NaN3) for 30 min at 4 °C 791
avoiding from light. To analysis macrophages apoptosis, cells were incubated with 792
Annexin V (BioLegend) in Annexin V Binding Buffer (BD Bioscience) at room 793
Page 36
36
temperature for 20 min in the dark, and then stained with PI (Propidium bromide) for a 794
maximum of three minutes. The samples were washed with cold FACS buffer and 795
analyzed with C6 (BD Bioscience). 796
Statistics. Adequate power was ensured when choosing the sample sizes. Data are 797
represented as the means ± SEM of at least three experiments. Statistical analyses are 798
performed using Graphpad Prism6 software, version 6. Statistical significance is 799
calculated using Student's two-tailed unpaired t-test for comparison between groups, and 800
using paired t-test for western blot and serum ELISA analysis. The log-rank test is used 801
for survival comparisons. NS, not significant (p>0.05); *P < 0.05, **P < 0.01. 802
Author Contributions: Y.L, and Y.Q. performed the majority of experiments and 803
statistical analysis with the help of H.C., L.G., F. Z., L. M., C. Z., X. Z., J. X., R.Z., L. H., 804
X.X., Y. Z., P.C. participated in part of the ubiquitination experiments. Y.H. generated 805
the interaction model of Hrd1 and Usp15. H.W., B.W., D.L., Y.L., Y.Q. G. Z., R. B., Y.H. 806
and R. H. designed the study. Y.L. and H.W. drafted the manuscript. 807
Acknowledgments: We thank Drs. ShaoCong Sun, Chen Wang, Bing Sun for providing 808
plasmids, Dr. Anning Lin for MEF cell lines, Dr. WH Fang for Salmonella typhimurium 809
(strain SL1344), Dr. Yulong He for Lysozyme M (LysM)-Cre+/+ mice, Dr. Ping Wang for 810
DUb-AMC reagents. We would like to the Core Facility of Chemical Biology and Core 811
Facility of Molecular Biology for technique help; thank the National Center for Protein 812
Science Shanghai for MS analysis and EM Data Collection. 813
This work was supported by grants from the Strategic Priority Research Program of 814
the Chinese Academy of Sciences (XDB19000000), the Ministry of Science and 815
Page 37
37
Technology of China (2016YFD0500207, 2016YFD0500407, and 2016YFC0905902), 816
National Natural Science Foundation of China (81825011, 81630043, 81571617, 817
81671572, 81571552, 81701569, 31700781, 8180060957), and the State Key Laboratory 818
of Cell Biology, SIBCB, CAS (SKL CBKF2013003). We thank the Genome Tagging 819
Project (GTP) Center, Shanghai Institute of Biochemistry and Cell Biology, CAS for 820
technical support. Dr. H. Wang is supported by the Hundred Talents Program of the 821
Chinese Academy of Sciences. 822
Competing interests: None declared. 823
Data availability: The original data that support the findings of this study are available 824
from the corresponding author upon request. Supplementary figures are available in the 825
Supplementary Information. 826
827
Page 38
38
Figure 1. RNAi screening identifies the ER-localised Hrd1 to positively regulate 828
inflammation in LPS-stimulated macrophages 829
(a) The relative amount of IL-6 concentrations in the siRNA screening plate that contains 830
LPS-treated mouse peritoneal macrophages (PEMs). (b) Electron microscopic images 831
from macrophages after incubation with the untreated beads (left panel) or LPS-coated 832
beads (right panel). Triangles indicate ER membrane. (c) Confocal microscopy analysis 833
of the ER in MEFs transfected with KDEL-mCherry before or after SL1344 infection. 834
Nucleus were labelled with DAPI. (d, e, f) The relative mRNA levels was checked by 835
qRT-PCR or cytokine concentrations was quantified by ELISA in PEMs transfected with 836
the control siRNA (N.C.) or Hrd1 siRNA for 72 hr, followed by LPS (1 μg/ml) 837
stimulation (d, f), SL1344 or E. coli infection (e). (g) qRT-PCR analysis of Il6 mRNA 838
levels in PMA-induced THP-1 cells or primary MEFs with or without LPS (1 μg/ml) 839
stimulation. THP-1 cells or primary MEFs were generated with a retrovirus transduction 840
system to stably overexpress GFP or Hrd1. (h) Luciferase (Luc) activity was measured in 841
HEK293T cells after transfected with the plasmids expressing NF-κB luciferase reporter, 842
renilla, GFP or Hrd1 without (left panel) or with (right panel) MyD88, TRAF6, IKKα, 843
IKKβ, p65 or TRIF for 24 hr. (i) Luciferase activity in HEK293T cells transfected with 844
the plasmids expressing NF-κB luciferase reporter, renilla, TRAF6, TRIF together with 845
the increasing doses of Hrd1. (j) Luciferase activity in HEK293T cells transfected with 846
the plasmids expressing NF-κB luciferase reporter, renilla, IKKβ together with GFP, WT 847
Hrd1, the transmembrane domain-deletion (ΔTM) or mitochondrial located (mito-Hrd1) 848
mutant. Similar expression levels of WT Hrd1, Hrd1 ΔTM and mito-Hrd1 were 849
confirmed by immunoblotting. (k) qRT-PCR analysis of Il6 mRNA levels in MEFs with 850
Page 39
39
or without LPS (1 μg/ml) stimulation. MEFs were generated with a retrovirus 851
transduction system to stably overexpress GFP, WT Hrd1 or the mitochondrial located 852
mutant (mito-Hrd1). Similar expression levels of Hrd1 and mito-Hrd1 were confirmed by 853
immunoblotting. Scale bar, 1 μm (b), 10 μm (c); ns, not significant (P> 0.05); *P <0.05; 854
**P< 0.01 (two-tailed Student’s t-test). Data are from 3 independent experiments (mean ± 855
SEM) (d-g, k) or representative of 3 independent experiments (b-c, h-j). n≥3. 856
857
Figure 2. Hrd1 KO macrophages specifically reduce TLR4-induced inflammation 858
and NF-κB activation 859
(a) Hrd1 KO efficiency was analyzed by immunoblotting with anti-Hrd1 antibody in 860
BMMs obtained from WT and cKO mice. (b-d) Immunoblot analysis of phosphorylated 861
(p-) IKKα/β and degradation of IκBα (b) or p65 in cytoplasmic or nuclear fraction (c) or 862
p-ERk, p-p38, p-Jnk (d) in WT or Hrd1 KO PEMs upon LPS stimulation at the indicated 863
time points. Tubulin was used as cytoplasmic protein control. Lamin-B was served as 864
nuclear protein control (c). (e, f) The relative mRNA levels of Il6, Tnfa, and Il1b (e) or 865
concentrations of IL-6 and TNF-α (f) in WT or Hrd1 KO PEMs or BMMs after treatment 866
with LPS, SL1344 or E. coli for 6 hr. (g) WT or Hrd1 KO PEMs were transfected with 867
the control siRNA/N.C. or two different p65 siRNA followed by LPS stimulation to 868
measure the mRNA levels of Il6 by qRT-PCR. (h) WT or Hrd1 KO BMMs or PEMs 869
were stimulated with CpG (5 μg/ml) for 6 hr, Poly(I:C) (10 μg/ml) for 3 hr to measure the 870
relative mRNA levels of Il6 and Ifnb. ns, not significant (P> 0.05); *P <0.05; **P< 0.01 871
(two-tailed Student’s t-test). Data are from 3 independent experiments (mean ± SEM) 872
(e-h) or representative of 3 independent experiments (a, b-d). n≥3. 873
Page 40
40
874
Figure 3. The E3 ligase activity of Hrd1 is critical for TLR4-induced NF-κB 875
activation independent of ERAD 876
(a) The NF-κB Luciferase readings were measured in HEK293T cells upon transfection 877
with plasmids expressing NF-κB luciferase reporter, renilla and MyD88, TRAF6 or TRIF, 878
and the GFP control, WT Hrd1 or C329S for 24 hr. Similar expression levels of WT Hrd1 879
or C329S were confirmed by immunoblotting. (b) PMA-induced THP-1 cells stably 880
overexpressing GFP control, WT Hrd1 or C329S were stimulated with LPS for 60 min, 881
immunoprecipitated with anti-IκBα antibody or rabbit IgG, followed by immunoblotting 882
with anti-IκBα antibody and anti-K48-Ubiquitin antibody to measure levels of 883
Lys48-linked polyubiquitinatin of IκBα. (c) IκBα degradation was checked by 884
immunoblotting in MEFs stably overexpressing WT Hrd1, the C329S or mito-Hrd1 885
mutant upon LPS stimulation. (d) Immunoblot analysis of p65 in the cytoplasmic or 886
nuclear fraction from PMA-induced THP-1 cells upon LPS stimulation for 30 min, which 887
stably overexpressed GFP, WT Hrd1 or C329S. (e) Confocal microscopy analysis of p65 888
nuclear translocation in MEFs before and after LPS stimulation, which stably 889
overexpressed GFP, WT Hrd1 or C329S. More than 300 cells in each sample were 890
measured to quantify p65 nuclear translocation rates. Scale bar, 20 μm. (f) WT or Hrd1 891
KO BMMs stably overexpressing with GFP, WT Hrd1 or C329S (left panel), or the 892
transmembrane domain deletion mutant ΔTM (right panel) were generated by retroviral 893
transduction system, which were treated with LPS to measure Il6 mRNA level. (g) WT or 894
Hrd1 KO PEMs were treated with LPS or tunicamycin for 6 hr, followed by qRT-PCR 895
analysis of the mRNA levels of Hspa5, Ddit3, Dnajb9 or Xbp1s. ns, not significant (P> 896
Page 41
41
0.05); *P <0.05; **P< 0.01. Data are from 3 independent experiments (mean ± SEM) (f-g) 897
or representative of 3 independent experiments (a-e). n≥3. Statistical significance is 898
calculated using Student's two-tailed unpaired t-test for comparison between groups (e). 899
900
Figure 4. The ER-localised Hrd1 directly binds Usp15 and regulates TLR4-induced 901
inflammation 902
(a) The Yeast two-hybrid data to measure Hrd1 interaction with Usp15. Repeated twice. 903
(b) GST pull-down assay showed that the recombinant GST-tagged full-length Hrd1 (not 904
the transmembrane domain-deletion (ΔTM) mutant) directly bound the recombinant 905
His-tagged Usp15 (upper lane). Lower panel showed the equimolar loading 906
concentrations of GST-Hrd1 (3 μg), GST-ΔTM (2.4 μg) or GST proteins (0.92 μg). (c) 907
PEMs were stimulated with LPS at the indicated time points to detect the endogenous 908
interaction between Hrd1 and Usp15 by immunoprecipitation with anti-Usp15 antibody, 909
followed by immunoblotting with anti-Hrd1 or anti-Usp15 antibodies. (d) The levels of 910
Usp15 were measured in ER fractions from resting or LPS-stimulated PEMs by 911
immunoblotting. Calnexin, Caspase3 and Aif (apoptotic inducing factor) were 912
respectively as the control proteins of ER, cytoplasmic, mitochondrial fractions. (e) 913
HEK293T cells were transfected with Flag-tagged WT Hrd1 or ΔTM and HA-tagged 914
Usp15, followed by immunoprecipitation with anti-HA antibody, then immunoblotting 915
with the indicated antibodies. (f) The relative mRNA levels of Il6, Tnfa and Ilb by 916
qRT-PCR (f), or levels of IκBα degradation by immunoblotting (g) were checked in 917
LPS-treated PEMs after transfected with the control siRNA or two different Usp15 918
siRNAs. The knocking down efficiencies of Usp15 and the protein levels of TLR4 were 919
Page 42
42
checked by immunoblotting. (h) WT or Hrd1 KO PEMs and BMMs were transfected 920
with the control siRNA or two different Usp15 siRNAs, followed by LPS stimulation to 921
check Il6 mRNA level by qRT-PCR. (i) PEMs were transfected with N.C. or two 922
different Usp15 siRNAs, followed by transfection with N.C. or two different p65 siRNAs. 923
After LPS stimulation, IL-6 concentrations were quantified by ELISA. (j) The NF-κB 924
luciferase activity was measured in HEK293T cells after transfection with plasmids 925
expressing NF-κB luciferase reporter, renilla, IKKβ, WT Usp15 or the C298A/C812A 926
mutant (M2), WT Hrd1 or C329S. Expression levels of Hrd1 or WT Usp15 and M2 were 927
shown by immunoblotting. ns, not significant (P> 0.05); *P <0.05; **P< 0.01 (two-tailed 928
Student’s t-test). Data are representative of 3 independent experiments (mean ± SEM) (f, 929
h, i) or representative of 3 independent experiments (a-e, g, j). n≥3. 930
931
Figure 5. Hrd1 promotes polyubiquitination of Lys21 in Usp15 and inactivates 932
Usp15 933
(a) HEK293T cells were transfected with His-tagged Ubiquitin (Ub), HA-tagged Usp15 934
(left and middle panels) or His-tagged Usp15 (right panel) together with GFP, 935
Flag-tagged WT Hrd1, the C329S or ΔTM mutants. Immunoprecipitation was performed 936
with anti-HA antibody or Ni-NTA to enrich Usp15, followed by immunoblotting with 937
anti-His or anti-Ub antibodies to measure Usp15 ubiquitination levels. Usp2cc (the 938
catalytic core of Usp2) was used to remove ubiquitination as a control. (b) Expression of 939
the E1, E2, HA-tagged Ub, the E3 WT or C329S Hrd1 and the control E3 HACE, 940
together with His-tagged Usp15 M2 were induced by IPTG (1 μM) in E. coli. His-tagged 941
Usp15 M2 was enriched by Ni-NTA followed by immunoblotting with anti-HA (Ub) 942
Page 43
43
antibody to measure Usp15 ubiquitination levels. (c, d) WT PEMs (c), or WT and Hrd1 943
KO PEMs (d) were stimulated with LPS and prepared for immunoprecipitation with 944
anti-Usp15 antibody followed by immunoblotting with anti-Ub antibody to check Usp15 945
ubiquitination levels. (e) HEK293T cells were transfected with Hrd1 together with 946
HA-tagged Usp15 or its mutants. Immunoprecipitation was performed with anti-HA 947
(Usp15) , followed by immunoblotting with anti-His (Ub) to check Usp15 ubiquitination 948
levels. (f, g) HEK293T cells were transfected with Myc-tagged WT or C329S Hrd1 949
together with Flag-tagged Usp15 or the K21R mutant. Enriched Usp15 and K21R were 950
obtained by immunoprecipitation with anti-Flag antibody to measure deubiquitinase 951
activity in an ubiquitin-AMC assay (f). Alternatively, enriched Usp15 and K21R was 952
incubated with K48-linked poly-ubiquitin chains (Ub2-16), followed by immunoblotting 953
with anti-Ub antibody to measure mono/poly-ubiquitin levels (g). (h) The NF-κB 954
luciferase activity was checked in HEK293T cells, which were transfected with NF-κB 955
luciferase reporter, renilla, IKKβ, Flag-tagged Hrd1, different doses of HA-tagged WT 956
Usp15 or K21R for 24 hr. (i) MEFs stably overexpressing GFP, WT Usp15 or K21R, 957
were infected with E.coli for 3 hr. IL-6 production at mRNA and protein levels was 958
analyzed by qRT-PCR and ELISA. ns, not significant (P> 0.05); *P <0.05; **P< 0.01 959
(two-tailed Student’s t-test). Data are from 3 independent experiments (mean ± SEM) (i) 960
or representative of 3 independent experiments (a-h). n≥3. 961
962
Figure 6. Hrd1 deficiency protects against LPS- and CLP- induced septic shock 963
(a, e) WT and Hrd1 cKO mice were i.p. injected with LPS (35 mg/kg) or subjected with 964
CLP operations to record survival rates. Each dot represents one mouse. (b-c, f-g) Serum 965
Page 44
44
IL-6 concentrations (b, f), Il6 mRNA expression in different organs (c, g) were measured 966
from WT and Hrd1 cKO mice 6hr after LPS (25 mg/kg) injection or CLP surgery. (d, h) 967
H&E staining of lungs was performed in WT and Hrd1 cKO mice 24hr after LPS (25 968
mg/kg) injection or CLP surgery. (i) WT or Hrd1 cKO PEMs were transfected with N.C. 969
or Usp15 siRNA, labeled with CFSE, and adoptively transferred into the recipient mice 970
that were previously injected with clodronate-containing liposomes to deplete 971
endogenous peritoneal macrophages. After the recipient mice were challenged in vivo 972
with LPS, CFSE+ PEMs were harvested to measure Il6 mRNA expression by qRT-PCR 973
(left panel). The KD efficiency of Usp15 was shown in the right panel. (j, k) WT and 974
Hrd1 cKO mice were i.v. injected with Poly(I:C) (100 μg/mouse) (j) or CpG (100 975
μg/mouse)(k), and expression of IFN-β and IL-6 in serum or at mRNA levels in organs 976
were measured. (l) The model. The ER-localised Hrd1 ubiquitinates and inactivates the 977
DUB Usp15 to promote TLR4-NF-κB induced inflammation independent of ERAD. 978
Scale bars in (d, h) 50 µm. ns, not significant (P> 0.05); *P< 0.05, **P< 0.01 and ***P< 979
0.001 (a-k, two-tailed Student’s t-test). Data are from 3 independent experiments (mean ± 980
SEM) of triplicate assays (b, c, f, g, i-k) or three independent experiments (d, h). n≥3. 981
982
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