AZI2基因敲除HEK293细胞

Damaged mitochondria are selectively eliminated in a process called mitophagy. PINK1 and Parkin amplify ubiquitin signals on damaged mitochondria, which are then recognized by autophagy adaptors to induce local autophagosome formation. NDP52 and OPTN, two essential mitophagy adaptors, facilitate de novo synthesis of pre-autophagosomal membranes near damaged mitochondria by linking ubiquitinated mitochondria and ATG8 family proteins and by recruiting core autophagy initiation components. The multifunctional serine/threonine kinase TBK1 also plays an important role in mitophagy. OPTN directly binds TBK1 to form a positive feedback loop for isolation membrane expansion. TBK1 is also thought to indirectly interact with NDP52; however, its role in NDP52-driven mitophagy remains largely unknown. Here, we focused on two TBK1 adaptors, AZI2/NAP1 and TBKBP1/SINTBAD, that are thought to mediate the TBK1-NDP52 interaction. We found that both AZI2 and TBKBP1 are recruited to damaged mitochondria during Parkin-mediated mitophagy. Further, a series of AZI2 and TBKBP1 knockout constructs combined with an OPTN knockout showed that AZI2, but not TBKBP1, impacts NDP52-driven mitophagy. In addition, we found that AZI2 at S318 is phosphorylated during mitophagy, the impairment of which slightly inhibits mitochondrial degradation. These results suggest that AZI2, in concert with TBK1, plays an important role in NDP52-driven mitophagy.

5-azacytidine-induced protein 2 (AZI2) is known to have a crucial role in antiviral innate immunity. This study aims to explore the roles of AZI2 in influenza-trigger pediatric pneumonia and its molecular mechanism. qPCR and immunoblotting assays were used to determine the levels of target genes and proteins. The lung infection mouse model was established by using PR8 H1N1 virus in AZI2 germline knockout (AZI2-/-) and wild-type (WT) mice. In addition, HEK293T cell-based luciferase reporter assays were used to investigate the regulatory effects of AZI2 on type I interferon. Immune precipitation and immunofluorescence staining were used to evaluate the interactions between AZI2 and TANK binding kinase 1 (TBK1). We observed an elevation in the expressions of IFN-I and AZI2 in peripheral blood mononuclear cells from the pneumonia patients with mild symptoms. Interestingly, AZI2 deficiency deteriorated the influenza-induced pathological symptoms in the lung as well as reduced the survival rate. It was further showed that AZI2 positively regulated the expressions of type I interferon, inflammatory cytokines, and IFN production-related genes. The molecular mechanism data revealed that AZI2 regulated the interactions between TBK1 and TANK. In summary, AZI2 positively regulates type I interferon production in influenza-induced pediatric pneumonia by promoting the interactions between TBK1 and TANK.