Stimulator of interferon genes (STING, also known as MITA, ERIS, or MPYS) is essential for host immune responses triggered by microbial DNAs. However, the regulatory mechanisms underlying STING-mediated signaling are not fully understood. We report here that, upon cytoplasmic DNA stimulation, the endoplasmic reticulum (ER) protein AMFR was recruited to and interacted with STING in an insulin-induced gene 1 (INSIG1)-dependent manner. AMFR and INSIG1, an E3 ubiquitin ligase complex, then catalyzed the K27-linked polyubiquitination of STING. This modification served as an anchoring platform for recruiting TANK-binding kinase 1 (TBK1) and facilitating its translocation to the perinuclear microsomes. Depletion of AMFR or INSIG1 impaired STING-mediated antiviral gene induction. Consistently, myeloid-cell-specific Insig1(-/-) mice were more susceptible to herpes simplex virus 1 (HSV-1) infection than wild-type mice. This study uncovers an essential role of the ER proteins AMFR and INSIG1 in innate immunity, revealing an important missing link in the STING signaling pathway.

A Huisgen 1,3-dipolar cycloaddition "click chemistry" was employed to immobilize azido sugars (mannose, lactose, alpha-Gal) to fabricate carbohydrate self-assembled monolayers (SAMs) on gold. This fabrication was based on preformed SAM templates incorporated with alkyne terminal groups, which could further anchor the azido sugars to form well-packed, stable, and rigid sugar SAMs. The clicked mannose, lactose, and alpha-Gal trisaccharide SAMs were used in the analysis of specific carbohydrate-protein interactions (i.e., mannose-Con A; ECL-lactose, alpha-Gal-anti-Gal). The apparent affinity constant of Con A binding to mannose was (8.7 +/- 2.8) x 10(5) and (3.9 +/- 0.2) x 10(6) M(-1) measured by QCM and SPR, respectively. The apparent affinity constants of lactose binding with ECL and alpha-Gal binding with polyclonal anti-Gal antibody were determined to be (4.6 +/- 2.4) x 10(6) and (6.7 +/- 3.3) x 10(6) M(-1), respectively by QCM. SPR, QCM, AFM, and electrochemistry studies confirmed that the carbohydrate SAM sensors maintained the specificity to their corresponding lectins and nonspecific adsorption on the clicked carbohydrate surface was negligible. This study showed that the clicked carbohydrate SAMs in concert with nonlabel QCM or SPR offered a potent platform for high-throughput characterization of carbohydrate-protein interactions. Such a combination should complement other methods such as ITC and ELISA in a favorable manner and provide insightful knowledge for the corresponding complex glycobiological processes.