Novel Toll/IL-1 Receptor Homologous Region Adaptors Act as Negative Regulators in Amphioxus TLR Signaling

This information is current as Jian Peng, Xin Tao, Rui Li, Jingru Hu, Jie Ruan, Ruihua of September 29, 2021. Wang, Manyi Yang, Rirong Yang, Xiangru Dong, Shangwu Chen, Anlong Xu and Shaochun Yuan J Immunol published online 31 August 2015 http://www.jimmunol.org/content/early/2015/08/30/jimmun

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published August 31, 2015, doi:10.4049/jimmunol.1403003 The Journal of Immunology

Novel Toll/IL-1 Receptor Homologous Region Adaptors Act as Negative Regulators in Amphioxus TLR Signaling

Jian Peng,*,1 Xin Tao,* Rui Li,* Jingru Hu,* Jie Ruan,* Ruihua Wang,* Manyi Yang,* Rirong Yang,* Xiangru Dong,* Shangwu Chen,* Anlong Xu,*,† and Shaochun Yuan*

Studies have shown that the basal chordate amphioxus possesses an extraordinarily complex TLR system, including 39 TLRs and at least 40 Toll/IL-1R homologous region (TIR) adaptors. Besides homologs to MyD88 and TIR domain-containing adaptor mol- ecule (TICAM), most amphioxus TIR adaptors exhibit domain architectures that are not observed in other species. To reveal how these novel TIR adaptors function in amphioxus Branchiostoma belcheri tsingtauense (bbt), four representatives, bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD, were selected for functional analyses. We found bbtTIRA to show a unique inhibitory role in amphioxus TICAM-mediated pathway by interacting with bbtTICAM and bbt receptor interacting protein 1b, whereas Downloaded from bbtTIRC specifically inhibits the amphioxus MyD88-dependent pathway by interacting with bbtMyD88 and depressing the polyubiquitination of bbt TNFR-associated factor 6. Although both bbtTIRB and bbtTIRD are located on endosomes, the TIR domain of bbtTIRB can interact with bbtMyD88 in the cytosol, whereas the TIR domain of bbtTIRD is enclosed in endosome, suggesting that bbtTIRD may be a redundant gene in amphioxus. This study indicated that most expanded TIR adaptors play nonredundant regulatory roles in amphioxus TLR signaling, adding a new layer to understanding the diversity and complexity of innate immunity at basal chordate. The Journal of Immunology, 2015, 195: 000–000. http://www.jimmunol.org/

oll-like receptors (TLRs) are a class of proteins that induction of type I IFN. The MyD88-dependent pathway is uni- recognize structurally conserved molecules derived from versal for all TLRs except TLR3, whereas the TRIF-dependent T microbes collectively referred to as pathogen-associated pathway is specifically used by TLR3 and TLR4 (3). When re- molecular patterns (1, 2). In mammals, upon ligand stimulation, cruited by TLR4, MyD88 requires MyD88 adaptor-like (MAL, the MyD88-dependent and Toll/IL-1R homologous region (TIR) also known as TIRPA), whereas TRIF needs TRIF-related adaptor domain-containing adaptor inducing IFN-b (TRIF)–dependent molecule (TRAM) as a partner (2, 4). The fifth adaptor SARM

pathways can be triggered, leading to activation of NF-kB and the plays an inhibitory role in the TRIF-dependent pathway (5, 6). by guest on September 29, 2021 As an important component of the innate immune system, mammalian TLR signaling is tightly regulated to avoid extreme *State Key Laboratory of Biocontrol, Guangdong Key Laboratory of Pharmaceutical Functional Genes, College of Life Sciences, Sun Yat-Sen University, Guangzhou immune responses and to maintain tissue homeostasis (7). At the 510275, People’s Republic of China; and †Beijing University of Chinese Medicine, receptor level, the TLR4 homolog, radioprotective 105-kDa pro- Beijing 100029, People’s Republic of China tein (RP105, also called CD180) inhibits LPS-TLR4 binding and 1 Current address: Department of Biotechnology, Guizhou Medical University, impairs LPS-mediated NF-kB activation. Suppression of tumori- Guiyang, Guizhou Province, People’s Republic of China. genicity 2–related protein also inhibits TLR4 signaling by com- Received for publication December 2, 2014. Accepted for publication August 3, 2015. peting for MyD88 binding (8, 9). Dominant-negative proteins This work was supported by National Basic Research Program (973) Project generated by stimulus-dependent alternative splicing are also 2013CB835303, National Natural Science Foundation of China Projects 31270018, regulators at the adaptor level. For example, an LPS-inducible 31470846, and 30730089, and New Star of Pearl River on Science and Technology splice variant of MyD88, MyD88s, is a dominant negative in- of Guangzhou Project 2014J2200017. hibitor of TLR/IL-1R–induced NF-kB activation (10). The splice The sequences presented in this article have been submitted to the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/genbank) un- variant IL-1R–associated kinase (IRAK)-1c and IRAK-M are der accession numbers KM288437, KM288438, KM288439, and KM288440. dominant-negative IRAK forms that play negative regulatory roles Address correspondence and reprint requests to Dr. Anlong Xu and Dr. Shaochun in Toll/IL-1R–induced inflammatory signaling (11, 12). In addi- Yuan, State Key Laboratory of Biocontrol, Department of Biochemistry, College of tion, posttranslation modification is widely used in negative Life Sciences, Sun Yat-Sen University, 135 Xingangxi Road, Guangzhou 510275, People’s Republic of China. E-mail addresses: [email protected] (A.X.) and feedback regulation of TLR signal (13, 14). For instance, activa- [email protected] (S.Y.) tion of the MyD88-dependent pathway leads to K63-linked The online version of this article contains supplemental material. polyubiquitination of TNFR-associated factor 6 (TRAF6) and Abbreviations used in this article: ABIN-1, A20 binding and inhibitor of NF-kB; the subsequent activation of the inhibitor of NF-kB kinase (IKK) ABL2, abelson murine leukemia viral oncogene homolog 2; bbt, Branchiostoma complex (15), whereas the K48-linked polyubiquitination of belcheri tsingtauense; bbtRIP1b, bbt receptor interacting protein 1b; CARD, caspase recruitment domain; Co-IP, coimmunoprecipitation; ER, endoplasmic reticulum; TRAF6 results in proteasomal degradation of TRAF6 and inhi- EST, expressed sequence tag; FPP, fluorescence protease protection; HEK, human bition of NF-kB (16). Because ubiquitination is a reversible pro- embryonic kidney; IKK, inhibitor of NF-kB kinase; IRAK, IL-1R–associated kinase; LTA, lipoteichoic acid; MyD88s, splice variant of MyD88; TICAM, TIR domain- cess, deubiquitination of TRAF6 is another effective feedback containing adaptor molecule; TIR, Toll/IL-1R homologous region; TRAF6, regulation of TLR signaling. TNFR-associated factor 6; TRAM, TRIF-related adaptor molecule; TRIF, TIR Compared with vertebrates, basal deuterostomes possess greatly domain-containing adaptor inducing IFN-b. expanded TLR repertoires. For example, the echinoderm sea urchin Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 has 222 TLRs and up to 26 potential TIR adaptors, whereas the

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1403003 2 TIR ADAPTORS NEGATIVELY REGULATE TLR PATHWAY IN AMPHIOXUS cephalochordate amphioxus possesses at least 39 TLRs and .40 For the NF-kB–specific inhibitor helenalin-treated experiments, the TIR adaptors (17). Among these adaptors, amphioxus MyD88, adult amphioxus were separated into three groups and treated with: 1) TRAM-like [also known as amphioxus TIR domain-containing DMSO as a negative control, 2) the LPS and LTA (1 mg/ml) mixtures, or 3) helenalin administered before inoculation with the LPS and LTA mix- adaptor molecule (TICAM)], and SARM have been well studied. tures (15 ml per animal). The animals were cultured in separate tanks, and Amphioxus MyD88 was shown to mediate the activation of NF- the intestines from five individuals were collected at 3 h postinjection as kB through its middle and death domains, whereas TICAM was a single sample. shown to mediate the most primitive TRIF-dependent activation The following primer pairs were used: BbtTIRA-F: 59-GGCTATCA- GGCTCTACGGACTTC-39, BbtTIRA-R: 59-CGCTTGACTTGCTGTTGC- of NF-kB (18–20). Amphioxus SARM plays inhibitory roles in TTGT-39; BbtTIRB-F: 59-TGGTCTACTTCGGTGGCTTGGA-39, BbtTIRB-R: both MyD88- and TICAM-dependent pathways by interacting 59-CGGCTCATCGCTCTGTTCATCTTG-39;BbtTIRC-F:59-GTCATC- with MyD88, TRAF6, and TICAM (19, 21). Recently, ubiquitination GGTGTCCAAGTT-39,BbtTIRC-R:59-TCTCCTCCAGCATGTCAT-39; was shown to be essential in regulating the activation of amphi- BbtTIRD-F: 59-AGCCTTCTACTGGACATACCTGAG-39,BbtTIRD-R:59-CA- oxus NF-kB (22, 23). In addition to these TIR adaptors having CGCCACCGACAAGACATATC-39; Bbtb-actin-F: 59-CCCTGTGCTGC- TGACTGAG-39, Bbtb-actin-R: 59-ACACGCCATCTCCAGAATCC-39. homologies with their vertebrate counterparts, most amphioxus TIR adaptors exhibit novel protein domain architectures, such as Expression plasmids the TIR domain linked with the kinase domain and the TIR do- For the expression of bbtTIRA in HEK293T cells, PCR fragments main linked with the caspase recruitment domain (CARD). To encoding for aa 1–160, 161–620, 621–889, 890–1142, and 1–1142 of reveal how these novel TIR adaptors function in amphioxus TLR bbtTIRA were inserted into pCMV-Flag, pCMV-Myc, and pCMV-HA (Clontech) and designated bbtTIRA1, bbtTIRA4, bbtTIRA7, bbtTIRA9, signaling, we selected four representatives for functional analyses. and bbtTIRA-FL, respectively (Fig. 3B). For the expression of bbtTIRC in The study will not only aid in defining the relationship among HEK293T cells, PCR fragments encoding for aa 1–161, 162–302, and Downloaded from amphioxus TIR adaptors, but also will provide further knowledge 1–302 of bbtTIRC were inserted into pCMV-Flag, pCMV-Myc, and on the regulation of TLR signaling in vertebrates. pCMV-HA (Clontech) and designated bbtTIRC1, bbtTIRC2, and bbtTIRC- FL, respectively (Fig. 4D). For the expression of bbtTIRB and bbtTIRD in HEK293T cells, PCR fragments encoding for aa 1–493 of bbtTIRB and 1– Materials and Methods 429 of bbtTIRD were inserted into pCMV-Flag, pCMV-Myc, and pCMV- Animals and cells HA (Clontech) and designated bbtTIRB-FL and bbtTIRD-FL, respectively.

For the study of subcellular localization, full-length bbtMyD88, bbtTICAM, http://www.jimmunol.org/ Wild adult Chinese amphioxus Branchiostoma belcheri tsingtauense (bbt) bbt receptor interacting protein 1b (bbtRIP1b), and bbtTRAFs were were obtained from Qingdao, China. Human embryonic kidney (HEK) inserted into pEGFP-N1 (Clontech). To determine topologies of membrane 293T and Hela cells were grown in DMEM supplemented with 10% FCS proteins, we inserted full-length bbtTIRD into pEGFP-N1 and inserted and antibiotics. full-length bbtTIRB into pEGFP-C1 (Clontech) (Fig. 2B). PCR fragment encoding for mCherry was inserted into pcDNA3.1 (Invitrogen). PCR Cloning of bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD cDNAs fragments encoding for aa 1–864 of bbtTICAM fused with 39Flag tag was TIR adaptor orthologs were identified in the B. floridae genome. Based on inserted into a pcDNA3.0 vector (Invitrogen) and designated bbtTICAM- these sequences, partial sequences of bbtTIRA, bbtTIRB, bbtTIRC, and FL. Full-length bbtMyD88, bbtTRAF6, and bbtRIP1b were inserted into bbtTIRD were cloned from bbt intestinal cDNA by specific primer pairs a pcDNA3.0 vector (Invitrogen) fused with 39Flag and 39HA tag. For the derived from bfTIRA (gene ID in B. floridae is 78250), bfTIRB (gene ID endosomal and mitochondrial markers, the PCR fragment encoding for the by guest on September 29, 2021 in B. floridae is 89683), bfTIRC (gene ID in B. floridae is 89399), and endosomal marker protein CD63 and the mitochondrial marker PHB1 bfTIRD (gene ID in B. floridae is 69123). Subsequently, 59-RACE and were inserted into pEGFP-N1 vectors fused with GFP-tag, respectively. 39-RACE were performed according to the manufacturer’s protocol using For the endoplasmic reticulum (ER), the PCR fragment encoding for the ER a GeneRACE Kit (Invitrogen) for full-length sequence cloning. These marker ERM was inserted into a pEGFP-N1 vector fused with RFP-tag. sequences have been submitted to the National Center for Biotechnology Fluorescence protease protection Information database (http://www.ncbi.nlm.nih.gov/genbank/) and the accession numbers are KM288437 (bbtTIRA), KM288440 (bbtTIRB), Protein localization could be determined by fluorescence protease pro- KM288438 (bbtTIRC), and KM288439 (bbtTIRD), respectively. tection (FPP) assay (24) and passage of the Hela cells into cell chambers (Lab-Tek chambered cover glass). At 16 h postpassage, cells were trans- Identification and sequence analysis of TIR adaptor-like genes fected with plasmid coding for the bbtTIRB or bbtTIRD tagged with GFP in Chinese amphioxus (B. belcheri) and soluble fluorescent protein mcherry (Red) expression plasmid. After 20 h posttransfection, cell culture medium was removed and cells were The draft genome of B. belcheri and the related analysis tools can be washed three times for 1 min each in KHM (110 mM potassium acetate, accessed at: http://mosas.sysu.edu.cn/genome. The following Pfam ac- 2 mM MgCl2, 20 mM HEPES, pH 7.2) buffer at 25˚C. Then 20 mM cession numbers were obtained from http://pfam.sanger.ac.uk/Software/ digitonin (Sigma-Aldrich) in KHM buffer was added to the cells to Pfam: Ankyrin repeat, PF00023; Arm, PF00514; CARD, PF00619; permeabilize the plasma membrane. To determine the permeabilization Death, PF00531; DED, PF01335; Glycos_transf_1, PF00534; HEAT, situation after digitonin application, the red fluorescent of mcherry (Red) PF02985; Helicase_C, PF00271; LRR_1, PF00560; MBT, PF02820; NB- was tested with microscopy. When the red fluorescent disappeared, the ARC, PF00931; OAS1_C, PF10421; Pkinase_Tyr, PF07714; Pro_isomer- KHM buffer with digitonin was removed and cells were washed quickly ase, PF00160; Ras, PF00071; ResIII, PF04851; RIG-I_C-RD, PF11648; but thoroughly with KHM buffer. Then 4 mM of the protease trypsin (in SAM_1, PF00536; SEFIR, PF08357; TIR, PF01582; TPR_1, PF00515; KHM buffer) was added directly onto the cells, and images were imme- TSP_1, PF00090; WD40, PF00400. Domain and gene identification were diately taken in the fluorescence microscope to record whether GFP mainly performed with the HMMER2.0 plus SMART dataset (http://smart. fluorescent signals persist or disappear. embl-heidelberg.de). Previously unidentified protein architectures were Coimmunoprecipitation (Co-IP), immunofluorescence imaging, tran- validated by searching the expressed sequence tag (EST) and cDNA evi- sient transfection, and luciferase reporter assay were performed as previ- dence from the EST dataset of B. belcheri, as well as the EST dataset and ously described (21, 25). cDNA sets of B. floridae (Supplemental Table I). Acute immune challenges of adult amphioxus and real-time Results PCR Sequences and structure analyses of bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD A total of 15 ml per animal of LPS and lipoteichoic acid (LTA; 1 mg/ml) mixture in PBS was injected into the amphioxus coelom. The challenged Annotation of immune-related molecules in amphioxus B. floridae and unchallenged amphioxus were cultured in separate tanks as described has identified .40 TIR adaptors and ongoing domain shuffling in our previous study (20). Intestines from five individuals were collected at 2, 4, 8, 12, 24, 36, 48, and 72 h postinjection as a single sample. among these adaptors (17). Using another recently completed Intestines from five PBS-injected animals (15 ml per animal) were col- genome of amphioxus B. belcheri, we conducted a genomic lected concurrently as nonchallenged controls. survey of the TIR adaptor in B. belcheri andcomparedwith The Journal of Immunology 3 Downloaded from

FIGURE 1. Genomic sequence analysis of bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD and their domain topology. (A–D) The TIR domain, STYKc domain, and transmembrane regions were predicted by the SMART Web site (http://smart.embl-heidelberg.de/smart/set_mode.cgi?NORMAL=1). The TRAF6-binding motif [PxExx(Ac/Ar): Ar for aromatic residues, Ac for acidic residues, x for any residue]. Sequence analyses indicated that all bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD possess several conserved kB-binding motifs in the promoter region, and the reporter assays confirmed that the region containing some kB-binding sites upstream of the ATG of bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD is essential for the binding to bbtRel. For the reporter http://www.jimmunol.org/ assays, the increasing amounts of pGL3 basic vectors containing 2-kb genomic sequences upstream of the ATG of bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD were transfected into HEK293T cells in the presence of 50 ng HsP65 or bbtRel expression vector. Hsp65 indicates Homo sapiens p65. Data are shown as mean 6 SD of three samples per treatment, and values were considered significant when p , 0.05. Results were confirmed by at least three separate experiments. *p , 0.05, **p , 0.01.

B. floridae. We found a similar number of TIR adaptors in the two ogies with MyD88, TICAM, and SARM, some adaptors were species. In addition to several TIR adaptors that showed homol- shown to be similar to orphan vertebrate TIR genes, and most of by guest on September 29, 2021

FIGURE 2. Inhibitory efforts of bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD on the NF-kB activation mediated by bbtMyD88 or bbtTICAM and their distributions. (A) bbtTIRB and bbtTIRD colocalized with the endosome marker CD63. Immunofluorescence microscopy images taken from HeLa cells cotransfected with GFP-fused CD63, together with HA-tagged pCMV-bbtTIRB, bbtTIRD, and stained with anti-HA and Alexa Fluor 532 Ab. (B) The FPP results showed that bbtTIRB anchored to the endosome with its TIR domain degraded by protease trypsin, whereas the TIR domain of bbtTIRD was not affected. The HeLa cells were transfected with mCherry (Red) inserted into pcDNA3.1 and bbtTIRB inserted into pEGFP-C1 or bbtTIRD inserted into pEGFP-N1. Fluorescence microscopy images were taken before or after digitonin and trypsin application. Fluorescence microscopy images are repre- sentative of at least three independent experiments in which .80% of the Hela cells showed similar patterns. (C–E) HEK293T cells were cotransfected with NF-kB transcriptional luciferase reporter constructs, together with bbtMyD88 or bbtTICAM and (increasing amounts of) bbtTIRA, bbtTIRB, and bbtTIRC vectors as indicated. All reporter assays performed in HEK293T cells are shown as mean 6 SD of three samples per treatment, and values were considered significant when p , 0.05. Results were confirmed by at least three separate experiments. *p , 0.05. 4 TIR ADAPTORS NEGATIVELY REGULATE TLR PATHWAY IN AMPHIOXUS them contain novel domain recombination, such as TIR+Pkinase- conserved protein structures: the TIR domain and the STYKc Tyr, CARD+TIR, and death effector domain (DED)+CARD+TIR domain. The STYKc domain of bbtTIRA showed 30% amino (Supplemental Table I). To reveal how the novel TIR adaptors acid identity with mammalian Tyrosine- abelson function in amphioxus TLR signaling, we chose eight TIR novel murine leukemia viral oncogene homolog 2 (ABL2) (Fig. 1A, adaptors (CARD-TIR, B. floridae gene ID: 89399, 96757; TIR- Supplemental Fig. 1A). BbtTIRC encodes a polypeptide of 302 aa HEAT, B. floridae gene ID: 93247; TIR-Pkinase_Tyr, B. floridae with a conserved orphan TIR domain and two TRAF6-binding gene ID: 78250; TPR-TIR, B. floridae gene ID: 63858 and 68270; motifs (PxExx) in the C terminus (Fig. 1C) (26, 27). Bedsides, TIR, B. floridae gene ID: 69123 and 89683) with the following bbtTIRB encodes a polypeptide of 493 aa, and bbtTIRD encodes two characteristics for further cloning. First, they should have a polypeptide of 430 aa. Although both bbtTIRB and bbtTIRD novel domain architectures. Second, they should exist in both contain two transmembrane regions and a TIR domain, the TIR B. belcheri and B. floridae simultaneously and not experience gene domain of bbtTIRB is located at the N terminus, whereas that of duplication. Adaptors with these two features should be more sta- bbtTIRD is located at the C terminus (Fig. 1B, 1D). Further se- bilized and have more specialized functions. quence alignment showed that the characteristic sequence Box1, Unfortunately, just four of them with differential characteristics Box2, Box3, and the BB loop are well conserved in the TIR could be isolated from the B. belcheri tsingtauense intestine cDNA domains of these four adaptors (Supplemental Fig. 1B). library and designated as bbtTIRA (corresponding to B. floridae gene ID: 78250), bbtTIRB (B. floridae gene ID: 89683), bbtTIRC bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD are the target genes k (B. floridae gene ID: 89399), and bbtTIRD (B. floridae gene ID: of NF- B

69123), respectively, suggesting that the other four TIR adaptors RT-PCR was performed to determine the tissue distribution of Downloaded from may be pseudogenes or genes with inducible transcription. bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD, and results showed BbtTIRA encodes a polypeptide of 1143 aa with two highly that transcripts of all the tested genes are abundant in amphioxus http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. bbtTIRA interacted with bbtTICAM and bbtRIP1b. (A) The full-length of bbtTIRA colocalized with bbtTICAM in Hela cells. (B) Truncated and site-directed mutants of bbtTIRA inserted into pCMV-Myc vector and named as indicated. Lys635 was substituted for Arg635.(C) bbtTIRA1, bbtTIRA4, and bbtTIRA7, but not bbtTIRA9, depressed the activation of the NF-kB by bbtTICAM. (D) Co-IP results showed that bbtTIRA1, bbtTIRA4, and bbtTIRA7, but not bbtTIRA9, could interact directly with bbtTICAM. (E) bbtTIRA inhibited NF-kB activation mediated by bbtRIP1b in HEK293T cells. (F) Confocal microscopy showed that bbtTIRA colocalized with bbtRIP1b. (G) The Co-IP results showed that bbtTIRA1, bbtTIRA4, and bbtTIRA7, but not bbtTIRA9, interacted directly with bbtRIP1b. (H) Compared with wild type bbtTIRA, the bbtTIRA-mutant could depress the activation of the NF-kBby bbtTICAM in a low level. Immunofluorescence microscopy images are representative of at least three independent experiments, in which .80% of the Hela cells showed similar staining patterns. All reporter assays performed in HEK293T cells are shown as mean 6 SD of three samples per treatment, and values were considered significant when p , 0.05. Results were confirmed by at least three separate experiments. *p , 0.05. The Journal of Immunology 5 digestive system (Supplemental Fig. 1C), which is considered to To reveal whether bbtTIRA, bbtTIRB, and bbtTIRC are critical be the first defense line of amphioxus (25, 28). To further study for the activation of NF-kB or other immune-related transcription the immunological significance of bbtTIRA, bbtTIRB, bbtTIRC, factors, we conducted luciferase assays. Because of the lack of and bbtTIRD in adults, we performed real-time PCR analyses, and amphioxus cell lines at present, mammal cell lines were chosen. results showed the transcriptions of these TIR adaptors in am- phioxus intestines to be upregulated after challenge with LPS and LTA mixture (Supplemental Fig. 1D). In our previous study, we have demonstrated that bbtRel is the ancestor of the vertebrate class II NF-kB proteins (25) and can interact with kB motif. Moreover, the interaction between kB motif and bbtRel can be blocked by the NF-kB–specific inhibitor helenalin (25, 29). In this study, we further showed that the upregulation of bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD were inhibited in adult amphi- oxus, which are treated with helenalin before challenge with LPS and LTA mixture (Supplemental Fig. 1E). These results suggested that the transcription of bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD are tightly regulated by amphioxus NF-kB. To further investigate whether bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD are classical NF-kB target genes, we obtained the Downloaded from 2-kb genomic sequences upstream of the ATG of bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD and subjected them to the Transcription Element Search System prediction program (www. cbil.upenn.edu/cgi-bin/tess/tess) to determine whether these regions contain conserved NF-kB–binding motifs. All promoter

regions of bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD were http://www.jimmunol.org/ found to contain several conserved kB-binding sites (Fig. 1). Then the sequence including kB-binding sites of bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD was inserted into pGL3 basic reporter vector and cotransfected with bbtRel or human p65 ex- pression plasmid in HEK293T to reveal whether p65 can recog- nize these promoter sequences. Reporter assays showed that hsp65 or bbtRel can recognize the sequence including kB-binding sites of bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD, suggesting that these TIR adaptors are classical targets of amphioxus NF-kB by guest on September 29, 2021 signaling (Fig. 1). bbtTIRA inhibits the bbtTICAM-dependent pathway, whereas bbtTIRB and bbtTIRC affect the bbtMyD88-dependent pathway Because bbtTIRB and bbtTIRD are transmembrane proteins with their TIR domain locating at a distinct terminus, to investigate whether they have a specific subcellular location, we cotrans- fected them with three organelle markers. Results showed bbtTIRB and bbtTIRD to be colocalized with endosomes, but not with mitochondrial and ER when overexpressed in Hela cells (Fig. 2A, Supplemental Fig. 2A, 2B). To investigate whether TIR domains of bbtTIRB and bbtTIRD faced to the cytosol, or enclosed in the endosome, we used further FPP experiment. In the FPP procedures, the bbtTIRB or bbtTIRD expressed cells were first treated with cholesterol-binding digitonin and then with trypsin. Because the plasma membrane contains more cholesterol than intracellular organelles, the low concentration of cholesterol-binding digitonin can increase the permeability of FIGURE 4. bbtTIRB and bbtTIRC interacted with bbtMyD88 and cell membrane and allow trypsin get into cytosol, but not intra- inhibited its transcription stimulatory activity. (A) The full length of cellular organelles. Results showed that, when treated with bbtTIRB and bbtTIRC colocalized with bbtMyD88. Immunofluorescence trypsin, the fluorescence of GFP disappeared in GFP-tagged microscopy images conducted in Hela cells are representative of at least bbtTIRB-expressed cells, but was retained in GFP-tagged three independent experiments, in which .80% of the cells showed similar bbtTIRD-expressed cells. These results suggested that bbtTIRB staining patterns. (B and C) The Co-IP results showed that bbtTIRB and D anchored to the endosome with its TIR domain faced to the cy- bbtTIRC interacted directly with bbtMyD88, but not with bbtTICAM. ( ) Truncated mutants used in this study. (E) bbtTIRC2 inhibited NF-kB ac- tosol, whereas the TIR domain of bbtTIRD enclosed in the tivation to the same extent as seen with the full length, but bbtTIRC1 had endosome or ER. When the TIR domain enclosed in the endo- no effect. (F) The Co-IP results showed that the mutant bbtTIRC1 and some or ER, it may have no chance to interact with other bbtTIRC2 interacted directly with bbtMyD88. All reporter assays per- TIR-containing molecules, suggesting that bbtTIRD may be a formed in HEK293T cells, and data are shown as mean 6 SD of three redundant protein without signaling transduction activities in samples per treatment. Values were considered significant when p , 0.05. amphioxus TLR signaling (Fig. 2B). Results were confirmed by at least three separate experiments. *p , 0.05. 6 TIR ADAPTORS NEGATIVELY REGULATE TLR PATHWAY IN AMPHIOXUS

Results showed none to have an effect on the induction of type I Then four truncated mutants of bbtTIRA were constructed (Fig. IFN or on the activation of AP1 and NF-kB in HEK293T cells 3B) and luciferase assays showed that the TIR domain, middle (Supplemental Fig. 2C–E). Because MyD88-dependent and region, and STYKc domains of bbtTIRA attenuated the activa- TICAM-dependent pathways have been demonstrated in amphi- tion of NF-kB mediated by bbtTICAM (Fig. 3C). Co-IP assays oxus (18, 19), we further used luciferase assays to test whether confirmed that TIR domain, middle region, and STYKc domain bbtTIRA, bbtTIRB, and bbtTIRC are involved in amphioxus of bbtTIRA, but not the C-terminal region, can interact with MyD88- and TICAM-dependent signaling. Results showed that bbtTICAM (Fig. 3D). Because our previous study demonstrated bbtTIRA specifically inhibited the bbtTICAM-dependent activa- that the association of bbtRIP1b with bbtTICAM is a crucial tion of NF-kB (Fig. 2C), whereas bbtTIRB and bbtTIRC could step in the bbtTICAM-mediated activation of NF-kB(19),to attenuate the bbtMyD88-mediated activation of NF-kB in a dose- investigate whether bbtTIRA would interrupt the interaction dependent manner (Fig. 2D, 2E). between bbtTICAM and bbtRIP1b, we performed luciferase assays. Results showed that bbtTIRA attenuated the activation BbtTIRA interacts with bbtTICAM and bbtRIP1b to inhibit the of NF-kB mediated by bbtRIP1b (Fig. 3E). Signal analysis by k bbtTICAM-mediated NF- B activation confocal microscopy indicated that bbtRIP1b colocalized with To further assess how bbtTIRA affects the bbtTICAM-mediated bbtTIRA when overexpressed in Hela cells (Fig. 3F). Further- activation of NF-kB, we first performed confocal microscopy more, Co-IP assays showed that bbtTIRA could interact with to show the colocalization of bbtTIRA with bbtTICAM, but not bbtRIP1b through its N-terminal, middle region, and STYKc with bbtMyD88 in Hela cells (Fig. 3A, Supplemental Fig. 2F). domain (Fig. 3G). Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 5. bbtTIRC inhibited the NF-kB activation induced by bbtTRAF6. (A) HEK293T cells were cotransfected with NF-kB transcriptional lucif- erase reporter constructs, together with bbtTRAF6 and increasing amounts of bbtTIRC vectors as indicated. (B) The full length of bbtTIRC colocalized with bbtTRAF6. Immunofluorescence microscopy images are representative of at least three independent experiments, in which .80% of the Hela cells showed similar staining patterns. (C) The Co-IP results showed bbtTIRC to interact directly with bbtTRAF6. (D) bbtTIRC2 inhibited NF-kB activation to the same extent as seen with the full length, but mutant bbtTIRC1 had no effect. (E) Co-IP results showed that bbtTIRC2 interacted directly with bbtTRAF6. (F) Site- directed mutants used. The TRAF6-binding motif (PxExx(Ac/Ar): Ar for aromatic residues, Ac for acidic residues, x for any residue). (G) bbtTIRC-mutant did not interact with bbtTRAF6. (H) Compared with the wild-type bbtTIRC, activity of bbtTIRC-mutant was attenuated to 40%. (I) Compared with the wild-type bbtTIRC, bbtTIRC mutant almost did not depress the polyubiquitination of bbtTRAF6. All reporter assays performed in HEK293T cells, and data are shown as mean 6 SD of three samples per treatment. Values were considered significant when p , 0.05. Results were confirmed by at least three separate experiments. *p , 0.05. The Journal of Immunology 7

Sequence analysis of the STYKc domain of bbtTIRA and TRAF6, but not bbt NF-kB essential modulator and human NF-kB mammalian ABL2 showed ∼30% identity (Supplemental Fig. 1A), essential modulator (Supplemental Fig. 3A–D). Compared with leading to the question of whether this domain can mediate ty- wild-type bbtTIRC, the ability of the bbtTIRC-mutant to depress rosine phosphorylation to affect NF-kB activation. The inactive polyubiquitination of TRAF6 was also attenuated (Fig. 5I). Thus, mutation of Abl2-like kinase into c-Abl1, in which the Lys290 we suggest that bbtTIRC inhibited the TRAF6-mediated signaling critical for ATP binding was mutated to arginine, resulted in by depressing the polyubiquitination of TRAF6 (Fig. 6). a complete loss of kinase effect (30). Thus, site-directed muta- genesis of bbtTIRA was conducted by replacing the conserved Discussion residues 635K within the STYKc domain with 635R (Fig. 3B). Rudiment of amphioxus TLR signaling Compared with wild type bbtTIRA, reporter assays showed that Basal deuterostomes possess many more TIR adaptors than the the K635R mutant can suppress the NF-kB activation mediated by five seen in vertebrates. For example, the sea urchin possesses 26 bbtTICAM in a low level (Fig. 3H). Thus, it appears that the TIR potential TIR adaptors, including 4 MyD88-like, 15 SARM1-like, domain, middle region, and STYKc domain of bbtTIRA partici- and 7 orphan TIR genes (33). In comparison of the genomes be- pated in inhibiting the activation of NF-kB in the MyD88-indepen- tween B. floridae and B. belcheri, we found that the basal chor- dent signal pathway. date amphioxus possesses .40 TIR adaptor proteins, including BbtTIRC interacts with bbtMyD88 and bbtTRAF6 and homologs of MyD88, TICAM, and SARM; adaptors similar to depresses polyubiquitination of bbtTRAF6 orphan vertebrate TIR genes; and adaptors with novel domain recombination (17). The identification of both TLR and MyD88 Because bbtTIRB and bbtTIRC were shown to attenuate the Downloaded from in various evolutionary stages, and the functional interaction NF-kB activation mediated by bbtMyD88, confocal microscopy of amphioxus TLR with MyD88, suggests that the MyD88- was performed. Results showed both bbtTIRB and bbtTIRC to dependent pathway is conserved and is crucial for invertebrate colocalize with bbtMyD88, but not with bbtTICAM when over- TLR signaling (18, 33–35). Because no homolog of verte- expressed in Hela cells (Fig. 4A, Supplemental Fig. 2G). Since brate TICAM1 and TICAM2 was identified in invertebrates, the bbtTIRB contains two transmembrane regions, it is easily MyD88-independent pathway was long believed to be a vertebrate gathered and shows significant organelle location (Fig. 2A, http://www.jimmunol.org/ Supplemental Fig. 2A, 2B). However, bbtTIRC may be widely distributed in the cytosol. Thus, when bbtMyD88 was co-trans- fected with bbtTIRB or bbtTIRC, it showed distinct distribution. Co-IP assays further confirmed the direct interaction of bbtMyD88 with both bbtTIRB and bbtTIRC (Fig. 4B, 4C). To investigate which domain of bbtTIRC is responsible for its function, two truncated mutants, bbtTIRC1 (the N-terminal) and bbtTIRC2 (the C-terminal TIR domain with TRAF6-binding motifs), were con- structed for further analysis (Fig. 4D). Reporter and Co-IP assays by guest on September 29, 2021 showed that although bbtTIRC1 and bbtTIRC2 were both shown to interact with bbtMyD88, only bbtTIRC2 inhibited NF-kB ac- tivation mediated by bbtMyD88 in a dose-dependent manner (Fig. 4E, 4F). Because recruitment of TRAF6 to MyD88 complexes is a key step in the activation of NF-kB (31, 32), we performed reporter assays to investigate the effect of bbtTIRC on bbtTRAF6 activity. Results showed that bbtTIRC inhibits TRAF6-mediated NF-kB activation in a dose-dependent manner (Fig. 5A). More- over, bbtTIRC can interact and colocalize with bbtTRAF6 (Fig. 5B, 5C). As mentioned earlier, the analysis of the bbtTIRC se- quence revealed two TRAF6-binding motifs (PxExx) in the C terminus. To verify whether the activity of bbtTIRC depends on these two motifs, two truncated mutants (bbtTIRC1 and bbtTIRC2) were coexpressed with bbtTRAF6. Results showed that only bbtTIRC2, which contains two TRAF6 binding motifs, can interact with bbtTRAF6 and inhibit the TRAF6-mediated activation of NF-kB in a dose-dependent manner (Fig. 5D, 5E). To further reveal whether the inhibitory activity of bbtTIRC is due to these two TRAF6 binding motifs, we conducted site-directed mutagenesis by replacing the conserved residues 162PVE164 and 257PPE259 into 162AVA164 and 257APA259 (Fig. 5F). In con- trast with wild-type bbtTIRC, the bbtTIRC-mutant did not interact with bbtTRAF6 (Fig. 5G), and the capacity of the bbtTIRC-mutant to attenuate the activation of NF-kBmediatedbybbtTRAF6was FIGURE 6. Novel TIR adaptors play nonredundant roles in both MyD88-dependent and -independent signal pathways in amphioxus. reduced to 40% (Fig. 5H). bbtTIRA shows a unique inhibitory role in amphioxus MyD88-indepen- Because K63-linked autopolyubiquitination of TRAF6 is an- dent pathway by interacting with bbtTICAM and bbtRIP1b, whereas other crucial step in the activation of NF-kB, to investigate whether bbtTIRC specifically inhibits the amphioxus MyD88-dependent pathway bbtTIRC could affect the polyubiquitination of bbtTRAF6, we by interacting with bbtMyD88 and depressing the polyubiquitination of performed ubiquitination assays. Results showed that bbtTIRC bbtTRAF6. At the same time, their expressions are controlled by NF-kB, can depress the polyubiquitination of bbtTRAF6 and human causing an effective feedback regulation of amphioxus NF-kB signaling. 8 TIR ADAPTORS NEGATIVELY REGULATE TLR PATHWAY IN AMPHIOXUS innovation. However, a common ancestor of vertebrate TICAM1 domain of bbtTIRA is important for the regulation of NF-kB. and TICAM2 was identified in amphioxus and shown to specifi- Studies in vertebrates have shown that Abl1, an Abl2-like tyrosine cally activate NF-kB in an MyD88-independent manner (19), kinase, can mediate the phosphorylation of inhibitor of NF-kB a suggesting that the primitive MyD88-independent pathway has at Tyr305 and increase its stability (42), resulting in the inhibition established in basal chordate. Because vertebrate TICAM1 can of NF-kB. Because the STYKc domain in bbtTIRA is homologous induce the production of type I IFN and activate NF-kB, verte- with the of Abl2, further characterization of the brate MyD88-independent signaling was considered to be origi- relationships of bbtTIRA with inhibitor of NF-kB a or other nated from the primitive bbtTICAM1–NF-kB pathway (19) and substrates should help to define the distinct functions of bbtTIRA coevolved with the emergence of the IFN system and adaptive in anti-inflammation. immunity in vertebrates (36). In this study, we further showed that In conclusion, to our knowledge, this study provides the first amphioxus TLRs can selectively bind to bbtTICAM or bbtMyD88 evidence that the expanded TIR adaptors in basal chordate play to activate NF-kB (Supplemental Fig. 3E–G). Thus, although both nonredundant roles in the activation of NF-kB, adding a further bbtTICAM- and bbtMyD88-dependent pathways converge upon layer of complexity to amphioxus innate immunity, not only with the IKK complex to activate NF-kB in amphioxus, they may as- respect to the diversity of receptor recognition, but also in the sociate with distinct TLRs to activate NF-kB, which provides cytoplasmic regulation (Fig. 6). quick and amplified immune responses when amphioxus encounters infections. Disclosures In the genome of amphioxus, it is indicated that there are ∼39 The authors have no financial conflicts of interest. TLRs, which are far more than that of the vertebrate. Besides the Downloaded from previously characterized bbtSARM, which plays inhibitory roles on both bbtMyD88- and bbtTICAM-dependent pathways by in- References teracting with bbtMyD88 and bbtTICAM in amphioxus. In this 1. Yamamoto, M., and K. Takeda. 2010. Current views of toll-like receptor sig- naling pathways. 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