Targeting of pro-apoptotic TLR adaptor SARM to mitochondria: definition of the critical region and residues in the signal sequence.

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.

SARM inhibits both TRIF- and MyD88-mediated AP-1 activation.

SARM (sterile alpha- and armadillo-motif-containing protein), the fifth identified TIR (Toll-interleukin 1 receptor (IL-1R)) domain-containing adaptors in humans, downregulates NF-kappaB 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-beta)-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.

Beyond TLR Signaling­The Role of SARM in Antiviral Immune Defense, Apoptosis & Development.

SARM (Sterile alpha and armadillo motif-containing protein) is the recently identified TIR domain-containing cytosolic protein. Classified as a member of the TLR adaptor family, the multiple locations and functions of SARM (sometimes playing opposing roles), provoke an enigma on its biology. Although originally assumed to be a member of the TLR adaptor family (functioning as a negative regulator of TLR signaling pathway), latest findings indicate that SARM regulates signaling differently from other TLR adaptor proteins. Recent studies have highlighted the significant functional role of SARM in mediating apoptosis and antiviral innate immune response. In this review, we provide an update on the evolutionary conservation, spatial distribution, and regulated expression of SARM to highlight its diverse functional roles. The review will summarize findings on the known interacting partners of SARM and provide analogy on how they add new dimensions to the current understanding on the multifaceted roles of SARM in antiviral activities and apoptotic functions. In addition, we provide a future perspective on the roles of SARM in differentiation and development, with substantial emphasis on the molecular insights to its mechanisms of action.

UXT plays dual opposing roles on SARM-induced apoptosis.

Apoptosis is a vital defense mechanism for the clearance of infected cells. Ubiquitously expressed transcript (UXT), which exists in two isoforms (V1 and V2), interact with both apoptotic and cellular proteins. By yeast two-hybrid analysis, we found that UXT interacts with SARM (sterile α and HEAT armadillo motif-containing protein). Since SARM is a TLR adaptor which induces intrinsic apoptosis following immune activation, we were prompted to query whether UXT and SARM might co-regulate apoptosis. We found that the UXT isoforms elicit dual opposing regulatory effects on SARM-induced apoptosis; while UXT V1, co-expressed with SARM, caused a reduction in caspase 8 activity, UXT V2 strongly increased caspase 8 activity and enhanced SARM-induced apoptosis by activating the extrinsic pathway and depolarizing the mitochondria.

In Silico Prediction of Evolutionarily Conserved GC-Rich Elements Associated with Antigenic Proteins of Plasmodium falciparum.

The Plasmodium falciparum genome being AT-rich, the presence of GC-rich regions suggests functional significance. Evolution imposes selection pressure to retain functionally important coding and regulatory elements. Hence searching for evolutionarily conserved GC-rich, intergenic regions in an AT-rich genome will help in discovering new coding regions and regulatory elements. We have used elevated GC content in intergenic regions coupled with sequence conservation against P. reichenowi, which is evolutionarily closely related to P. falciparum to identify potential sequences of functional importance. Interestingly, ~30% of the GC-rich, conserved sequences were associated with antigenic proteins encoded by var and rifin genes. The majority of sequences identified in the 5' UTR of var genes are represented by short expressed sequence tags (ESTs) in cDNA libraries signifying that they are transcribed in the parasite. Additionally, 19 sequences were located in the 3' UTR of rifins and 4 also have overlapping ESTs. Further analysis showed that several sequences associated with var genes have the capacity to encode small peptides. A previous report has shown that upstream peptides can regulate the expression of var genes hence we propose that these conserved GC-rich sequences may play roles in regulation of gene expression.