Mitochondrial Ubiquitin Ligase MARCH5 Promotes TLR7 Signaling by Attenuating TANK Action

et al. (2011) Mitochondrial Ubiquitin Ligase MARCH5 Promotes TLR7 Signaling by Attenuating TANK Action. PLoS Pathog 7(5): e1002057. doi:10.1371/journal.ppat.1002057 Mitochondrial Ubiquitin Ligase MARCH5 Promotes TLR7 Signaling by Attenuating TANK Action He-Xin Shi. 0 Xing Liu. 0 Qiang Wang 0 Pei-Pei Tang 0 Xin-Yi Liu 0 Yu-Fei Shan 0 Chen Wang 0 Barbara Sherry, North Carolina State University, United States of America 0 Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences , Shanghai , China 1 www.plospathogens.org The signaling of Toll-like receptors (TLRs) is the host's first line of defense against microbial invasion. The mitochondrion is emerging as a critical platform for antiviral signal transduction. The regulatory role of mitochondria for TLR signaling remains to be explored. Here, we show that the mitochondrial outer-membrane protein MARCH5 positively regulates TLR7 signaling. Ectopic expression or knockdown of MARCH5 enhances or impairs NF-kB-mediated gene expression, respectively. MARCH5 interacts specifically with TANK, and this interaction is enhanced by R837 stimulation. MARCH5 catalyzes the K63linked poly-ubiquitination of TANK on its Lysines 229, 233, 280, 302 and 306, thus impairing the ability of TANK to inhibit TRAF6. Mislocalization of MARCH5 abolishes its action on TANK, revealing the critical role of mitochondria in modulating innate immunity. Arguably, this represents the first study linking mitochondria to TLR signaling. - Funding: This work was supported by grants from Ministry of Science and Technology of China (2007CB914504, 2010CB529703, 2011CB910900); National Natural Science Foundation of China (30900762, 31030021); Ministry of Science and Technology of Shanghai (09XD1404800, 09ZR1436800). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Germline-encoded pattern recognition receptors (PRRs) play pivotal roles in sensing a wide range of invading pathogens, via recognizing conserved microbial signature molecules (PAMPs, pathogen associated molecular patterns). As molecular switches that register microbial infection, these receptors promptly initiate innate immune responses and subsequently prime the adaptive immune system to eliminate the pathogens [1,2,3]. Toll-like receptors (TLRs) are one major class of PRRs. To date, 13 members of the TLR family have been identified in mammals, of which TLR4 recognizes lipopolysaccharide (LPS) from gram-negative bacteria, and TLR7 senses viral single strand RNA (ssRNA) [2]. Both TLR4 and TLR7 can induce the robust expression of proinflammatory cytokines and type I interferons (IFNs). Recent years have witnessed an incredible gain in knowledge about the TLR signaling cascades. For example, in response to stimuli, TLR7 triggers the recruitment of MyD88 via its Tollinterleukin 1 receptor (IL-1R) homology (TIR) domain, which in turn recruits IRAK4 and IRAK1. IRAK4 then activates IRAK-1 by phosphorylation. As a result, the IRAKs dissociate from MyD88 and interact with TRAF6, a ubiquitin E3 ligase [4,5,6]. Together with Ubc13 and Uev1A, TRAF6 catalyzes the formation of lysine 63 (K63)-linked poly-ubiquitin chains, which serve as the anchoring platform for a protein complex that includes TRAF6, TRAF3, IKKa, and IRF7, leading ultimately to the induction of type I IFNs and ISGs (interferon-inducible genes) [7,8]. In the meantime, TAK1 is recruited to the TRAF6 protein complex, resulting in the activation of NF-kB and the induction of proinflammatory cytokines [9]. Given the critical roles of TLR signaling in innate immunity, multiple layers of stringent regulations are employed to ensure that the strength and duration of the TLR signal is appropriate for any given immune response. Several proteins have been demonstrated to modulate the TLR signaling pathways, such as A20, CYLD, IRAKM, ST2, SIGIRR and SOCS1 [10,11,12,13,14,15,16]. Importantly, TANK (also called I-TRAF) was initially characterized as a TRAF binding protein [17,18]. Recently, the in vivo function of TANK has been further clarified. Surprisingly, TANK is not essential for interferon induction and instead is a potent negative regulator for TLR-mediated induction of proinflammatory cytokines [19]. How TANK specifically modulates NF-kB signaling upon TLR activation remains to be determined. Mitochondria are rapidly emerging as important platforms for intracellular antiviral signaling. MAVS (also known as IPS-1/ VISA/Cardif) is the first mitochondrial protein identified as a critical component of the RIG-I/MDA5 signaling pathway [20]. Following this, several more mitochondrial proteins have been implicated in modulating this same antiviral signaling pathway, such as NLRX1, STING (also known as MITA) and MFN1 [21,22,23,24]. We have recently discovered that the mitochondrial outer-membrane receptor TOM70 mediates IRF3 activation downstream of MAVS [25,26,27]. Notably, whether any mitochondrial protein(s) can regulate TLR signaling remains an open question. It was recently reported that two ubiquitin E3 ligases constitutively express on mitochondria, MARCH5/MITOL/RNF153 and GIDE (Growth Inhibition and Death E3 Ligase). MARCH family proteins are characterized by harboring a RING-CH domain and multiple trans-membrane domains. Interestingly, many RING proteins In 2005, MAVS was characterized as the critical adaptor protein for the signal transduction of RIG-I-like receptors (RLRs). This provided the first link between mitochondria and the intracellular antiviral defense system. From then on, exploring the potential functions of novel mitochondrial proteins in microbe-host interactions became a rapidly expanding frontier. Notably, it remains unknown whether mitochondrial proteins can directly regulate TLR signaling. Here, we demonstrate that the mitochondrial protein MARCH5 positively modulates TLR7 signaling. Our study reveals that MARCH5 is a novel E3 ubiquitin ligase and catalyzes the K63-linked poly-ubiquitination of TANK. This modification releases the inhibitory effects of TANK on TRAF6. Arguably, this represents the first study linking mitochondria to TLR signaling, shedding new light on the role of mitochondria in the proinflammatory response. function as E3 ubiquitin ligases. Some of the MARCH proteins (MARCH1, MARCH8) appear to either directly or indirectly modulate immune functions by controlling the surface turnover of immune regulatory molecules on the plasma membrane [28]. MARCH5 (also named MARCH-V/MITOL/RNF153) was recently identified as a new member in the MARCH family [29,30]. Preliminary characterization uncovered MARCH5 as a novel mitochondrial protein. Until recently, little was known about the potential function of MARCH (...truncated)