The microphthalmia-associated transcription factor family (MiT family) proteins are evolutionarily conserved transcription factors that perform many essential biological functions. In mammals, the MiT family consists of MITF (microphthalmiaassociated transcription factor or melanocyte-inducing transcription factor), TFEB (transcription factor EB), TFE3 (transcription factor E3), and TFEC (transcription factor EC). These transcriptional factors belong to the basic helix-loophelix-leucine zipper (bHLH-LZ) transcription factor family and bind the E-box DNA motifs in the promoter regions of target genes to enhance transcription. The best studied functions of MiT proteins include lysosome biogenesis and autophagy induction. In addition, they modulate cellular metabolism, mitochondria dynamics, and various stress responses. The control of nuclear localization via phosphorylation and dephosphorylation serves as the primary regulatory mechanism for MiT family proteins, and several kinases and phosphatases have been identified to directly determine the transcriptional activities of MiT proteins. In different immune cell types, each MiT family member is shown to play distinct or redundant roles and we expect that there is far more to learn about their functions and regulatory mechanisms in host defense and inflammatory responses.

Nucleotide-sensing Toll-like receptors (TLRs), such as TLR3, 7, and 9, reside in the endolysosomal compartments to avoid activation by host DNA or RNA. Proper intracellular localization and signaling by these TLRs depend on the physical interaction with UNC93B1. Specific amino acid residues in UNC93B1 have been identified to affect the functions of endosolysomal TLRs. However, it is largely unknown how UNC93B1 differentially regulates individual TLRs. Protein N-glycosylation controls folding, maturation, stability, localization and protein-interaction of target glycoproteins. In this study, we examined whether the N-glycosylation of UNC93B1 affects its properties and molecular function. Using mutagenesis, we found that the N251 and N272 residues of UNC93B1 become N-glycosylated. We expressed wild type (WT) and N-glycosylation-defective UNC93B1 mutants (N251Q and N272Q) in UNC93B1-deficient cells and found that signaling of TLR9, but not that of other TLRs, were defective in UNC93B1 N272Q-expressing cells. The N272Q mutation did not affect the protein stability, localization, interaction of UNC93B1 and TLRs, nor did it prevent the CpG DNA-binding of TLR9. Nonetheless, upon CpG DNA stimulation, the recruitment of MyD88 to TLR9 was significantly inhibited in UNC93B1 N272Q-expressing cells compared to WT cells. Consequently, poor phosphorylation and degradation of I_B were observed in UNC93B1 N272Q-expressing cells. Combined, our data show that N-glycosylation of UNC93B1 specifically regulates TLR9 signaling by modulating the recruitment of MyD88 to TLR9.

Toll-like receptors (TLRs) play critical roles in the first line of host defense against pathogens through recognition of pathogen-associated molecular patterns and initiation of the innate immune responses. The proper localization of TLRs in specific subcellular compartments is crucial for their ligand recognition and downstream signaling to ensure appropriate responses against pathogens while avoiding erroneous or excessive activation. Several TLRs, including TLR7 and TLR9 but not TLR4, depend on UNC93B1 for their proper intracellular localization and signaling. Accumulating evidence suggest that UNC93B1 differentially regulates its various client TLRs, but the specific mechanisms by which UNC93B1 controls individual TLRs are not well understood. Protein N-glycosylation is one of the most frequent and important post-translational modification that occurs in membrane-localized or secreted proteins. UNC93B1 was previously shown to be glycosylated at Asn251 and Asn272 residues. In this study, we investigated whether N-glycosylation of UNC93B1 affects its function by comparing wild type and glycosylation-defective mutant UNC93B1 proteins. It was found that glycosylation of Asn251 and Asn272 residues can occur independently of each other and mutation of neither N251Q or N272Q in UNC93B1 altered expression and localization of UNC93B1 and TLR9. In contrast, CpG DNA-stimulated TLR9 signaling was severely inhibited in cells expressing UNC93B1(N272Q), but not in cells with UNC93B1(N251Q). Further, it was found that glycosylation at Asn272 of UNC93B1 is essential for the recruitment of MyD88 to TLR9 and the subsequent downstream signaling. On the other hand, the defective glycosylation at Asn272 did not affect TLR7 signaling. Collectively, these data demonstrate that the glycosylation at a specific asparagine residue of UNC93B1 is required for TLR9 signaling and the glycosylation status of UNC93B1 differently affects activation of TLR7 and TLR9.

Tetraspanin family proteins form a rigid membrane structure called tetraspanin-enriched microdomain (TEM) on the plasma membrane and regulate multiple cellular processes. CD82, a tetraspanin protein, is highly expressed in various immune cells, but its role in immune responses has been poorly understood. In this study, we investigated the role of CD82 in T cells using CD82 knock-out (KO) mice. At steady state, CD82 KO mice had less memory CD8+ T cells in secondary lymphoid organs compared to wild type mice, which became more pronounced in aged mice. Upon in vitro T cell receptor (TCR) stimulation, CD82 KO CD8+ T cells showed defects in phosphorylation of TCR downstream signaling molecules, activation marker expression, proliferation, and cytokine production. By confocal immunofluorescence imaging, we found that TCR stimulation-induced translocation of the microtubule organizing center and formation of the immunological synapse was impaired in CD82 KO T cells. CD82 seems to recruit T cell polarity-regulating molecules such as Scribble via the PDZ domain-binding motif located in its C-terminus and mediate the T cell polarization. In association with the defective CD8+ T cell activation caused by CD82 deficiency, CD82 KO CD8+ T cells show less efficient anti-tumor immune responses than wild-type cells when transferred to tumor-bearing mice. Collectively, our study demonstrates that CD82 mediates the T cell polarization and is required for the optimal CD8+ T cell effector function.