SARM (sterile a-and armadillo-motif-containing protein), the fifth identified TIR (Tollinterleukin 1 receptor (IL-1R)) domain-containing adaptors in humans, downregulates NF-jB and IRF3 (interferon-regulatory factor 3)-mediated TLR3 and TLR4 signaling. SARM was characterized as a negative regulator of the TRIF (TIR-domain-containing adaptor protein inducing IFN-b)-dependent pathway via its interaction with TRIF. However, the precise mechanism of action of SARM remains unclear. Here, we demonstrate that SARM inhibits MAPK activation in human embryonic kidney 293 cells, and U937 cells. Both the TRIF-and MyD88-mediated, as well as basal MAPK activity, were repressed, indicating that SARM-mediated inhibition may not be exclusively directed at TRIF or MyD88, but that SARM may also directly inhibit MAPK phosphorylation. The MAPK inhibition effect was verified by RNAi, which increased the basal level of AP-1. Furthermore, LPS challenge upregulated SARM at both the mRNA and protein levels. Finally, we provide evidence to show that truncated SARM changes its subcellular localization, suggesting the importance of the N-terminal and sterile alpha motif domains in the autoregulation of SARM activity.Key words: Activator protein 1 inhibition . MAPK . N-terminal polybasic and glycine-rich region motif . Sterile a-and armadillo-motif-containing protein . TLR Supporting Information available online IntroductionThe transmembrane TLR play a vital role in initiating innate immunity against pathogens [1]. To date, 13 members of the TLR family have been identified in mammals [2], all of which contain an intracellular TIR (Toll-interleukin 1 receptor (IL-1R)) domain [3]. TLR are a family of PRR which recognize PAMP. Different TLR recognize different PAMP, such as LPS (a ligand for TLR4) or double-stranded viral RNA (a ligand for TLR3). After activation by PAMP, TLR transduce specific immune-related signals to the nucleus via transcription factors such as NF-kB, interferonregulatory factor 3 (IRF3) and activator protein 1 (AP-1), which in turn induces pro-inflammatory mediators, including type I interferons, chemokines and cytokines [4].TLR exert their functions via a family of five TIR domaincontaining adaptor proteins: MyD88 (myeloid differentiation à These authors have contributed equally to this work. primary-response gene 88), Mal (MyD88-adaptor-like protein), TRIF (TIR-domain-containing adaptor protein inducing IFN-b), TRAM (TRIF-related adaptor molecule) and SARM (sterile a-and armadillo-motif-containing protein). MyD88, Mal, TRIF and TRAM all activate the TLR signaling pathways. All TLR except TLR3 recruit MyD88 to their cytoplasmic TIR domain to mediate innate immune signaling [5,6]. Mal is required by TLR2 and TLR4 to bridge the recruitment of MyD88 [7][8][9][10]. TRIF mediates TLR3 signaling and TLR4-induced MyD88-independent pathway, such as delayed NF-kB activation [11][12][13]. The interaction between TRIF and TLR4 is mediated by TRAM [14][15][16].As a newly discovered member of the TLR-adaptor family, the function of S...

The fifth and the most well-conserved member of the TLR (Toll-like receptor) adaptor, SARM (sterile α- and HEAT/armadillo-motif-containing protein), has been reported to be an important mediator of apoptosis. However, the exact cellular localization of SARM with respect to its role is unclear. In the present study we show that SARM specifically co-localizes with mitochondria. Endogenous SARM is mainly found in the mitochondria. We demonstrate that the N-terminal 27 amino acids (S27) of SARM, which is hydrophobic and polybasic, acts as a mitochondria-targeting signal sequence, associating SARM to the mitochondria. The S27 peptide has an inherent ability to bind to lipids and mitochondria. This sequence effectively translocates the soluble EGFP (enhanced green fluorescence protein) reporter into the mitochondria. Positioning S27 downstream of the EGFP abrogates its mitochondria-targeting ability. Transmission electron microscopy confirms the ability of S27 to import EGFP into the mitochondria. Importantly, by mutagenesis study, we delineated the specificity of the mitochondria-targeting ability to the arginine residue at the 14th position. The R14A SARM mutant also showed reduced apoptotic potential when compared with the wild-type. Taken together, S27, which is a bona fide signal sequence that targets SARM to the mitochondria, explains the pro-apoptotic activity of SARM.

Following acute-phase infection, activated T cells are terminated to achieve immune homeostasis, failure of which results in lymphoproliferative and autoimmune diseases. We report that sterile α- and heat armadillo-motif-containing protein (SARM), the most conserved Toll-like receptors adaptor, is proapoptotic during T-cell immune response. SARM expression is significantly reduced in natural killer (NK)/T lymphoma patients compared with healthy individuals, suggesting that decreased SARM supports NK/T-cell proliferation. T cells knocked down of SARM survived and proliferated more significantly compared with wild-type T cells following influenza infection in vivo. During activation of cytotoxic T cells, the SARM level fell before rising, correlating inversely with cell proliferation and subsequent T-cell clearance. SARM knockdown rescued T cells from both activation- and neglect-induced cell deaths. The mitochondria-localized SARM triggers intrinsic apoptosis by generating reactive oxygen species and depolarizing the mitochondrial potential. The proapoptotic function is attributable to the C-terminal sterile alpha motif and Toll/interleukin-1 receptor domains. Mechanistically, SARM mediates intrinsic apoptosis via B cell lymphoma-2 (Bcl-2) family members. SARM suppresses B cell lymphoma-extra large (Bcl-xL) and downregulates extracellular signal-regulated kinase phosphorylation, which are cell survival effectors. Overexpression of Bcl-xL and double knockout of Bcl-2 associated X protein and Bcl-2 homologous antagonist killer substantially reduced SARM-induced apoptosis. Collectively, we have shown how T-cell death following infection is mediated by SARM-induced intrinsic apoptosis, which is crucial for T-cell homeostasis.