TNFAIP3 promotes survival of CD4 T cells by restricting MTOR and promoting autophagy

<p>Autophagy plays important roles in metabolism, differentiation, and survival in T cells. TNFAIP3/A20 is a ubiquitin-editing enzyme that is thought to be a negative regulator of autophagy in cell lines. However, the role of TNFAIP3 in autophagy remains unclear. To determine whether TNFAIP3 regulates autophagy in CD4 T cells, we first analyzed <i>Tnfaip3</i>-deficient naïve CD4 T cells in vitro. We demonstrated that <i>Tnfaip3</i>-deficient CD4 T cells exhibited reduced MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) puncta formation, increased mitochondrial content, and exaggerated reactive oxygen species (ROS) production. These results indicate that TNFAIP3 promotes autophagy after T cell receptor (TCR) stimulation in CD4 T cells. We then investigated the mechanism by which TNFAIP3 promotes autophagy signaling. We found that TNFAIP3 bound to the MTOR (mechanistic target of rapamycin) complex and that <i>Tnfaip3</i>-deficient cells displayed enhanced ubiquitination of the MTOR complex and MTOR activity. To confirm the effects of enhanced MTOR activity in <i>Tnfaip3</i>-deficient cells, we analyzed cell survival following treatment with Torin1, an MTOR inhibitor. <i>Tnfaip3</i>-deficient CD4 T cells exhibited fewer cell numbers than the control cells in vitro and in vivo. In addition, the impaired survival of <i>Tnfaip3</i>-deficient cells was ameliorated with Torin1 treatment in vitro and in vivo. The effect of Torin1 was abolished by <i>Atg5</i> deficiency. Thus, enhanced MTOR activity regulates the survival of <i>Tnfaip3</i>-deficient CD4 T cells. Taken together, our findings illustrate that TNFAIP3 restricts MTOR signaling and promotes autophagy, providing new insight into the manner in which MTOR and autophagy regulate survival in CD4 T cells.</p

Novel polyubiquitin imaging system, PolyUb-FC, reveals that K33-linked polyubiquitin is recruited by SQSTM1/p62

<p>Ubiquitin chains are formed with 8 structurally and functionally distinct polymers. However, the functions of each polyubiquitin remain poorly understood. We developed a polyubiquitin-mediated fluorescence complementation (PolyUb-FC) assay using Kusabira Green (KG) as a split fluorescent protein. The PolyUb-FC assay has the advantage that monoubiquitination is nonfluorescent and chain-specific polyubiquitination can be directly visualized in living cells without using antibodies. We applied the PolyUb-FC assay to examine K33-linked polyubiquitin. We demonstrated that SQSTM1/p62 puncta colocalized with K33-linked polyubiquitin and this interaction was modulated by the ZRANB1/TRABID-K29 and -K33 linkage-specific deubiquitinase (DUB). We further showed that the colocalization of K33-linked polyubiquitin and MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) puncta was impaired by SQSTM1/p62 deficiency. Taken together, these findings provide novel insights into how atypical polyubiquitin is recruited by SQSTM1/p62. Finally, we developed an inducible-PolyUb-FC system for visualizing chain-specific polyubiquitin. The PolyUb-FC will be a useful tool for analyzing the dynamics of atypical polyubiquitin chain generation.</p