1

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

2

*Contributed equally; #Corresponding author: hongyanwang@sibcb.ac.cn; 30

weibinwhy@shu.edu.cn; coryhu@sibcb.ac.cn 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

3

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

4

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

5

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

6

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

7

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

8

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

9

(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

10

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

11

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

12

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

13

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

14

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

15

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

16

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

17

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

18

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

19

(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

20

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

21

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

22

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

23

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

24

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

25

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

26

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

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

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

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

30

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

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

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

33

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

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

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

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

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

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

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

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

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

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

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

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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