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. 2022 Feb 14;8:63. doi: 10.1038/s41420-022-00861-5

Fig. 5. Sirt5-dependent desuccinylation of OPTN promotes autophagic flux.

Fig. 5

A Endogenous OPTN bound to endogenous Sirt5 in 293T cells. Endogenous Co-IP of OPTN and Sirt5 was performed as indicated in the lysates of 293T cells, and the association was examined by western blotting. B, C Tagged Sirt5 interacted with and tagged OPTN. 293T cells were transfected with the indicated plasmids, and the OPTN-Sirt5 association was examined by Co-IP and western blotting. D Exogenous OPTN colocalized with Sirt5 in 293T cells. 293T cells were transfected with lentiviruses encoding OPTN-Cherry and Sirt5-Zsgreen. Scale bar = 5 μm. E Sirt5 desuccinylated endogenous OPTN. 293T cells were transfected with the Sirt5-HA plasmid, followed by IP-OPTN. The succinylation level of OPTN was determined by western blotting. F The OPTN K108E mutant abolished the interaction with Sirt5. 293T cells overexpressing Sirt5 were transfected with the indicated plasmids, and their interaction with OPTN was analyzed by IP and western blotting. G, I The OPTN K108E mutant abolished Sirt5-induced autophagic flux activation of OPTN in R28 cells and primary rRGCs. Representative images of primary rRGCs and R28 cells transfected with mCherry-GFP-LC3B lentivirus following transfection with the indicated plasmids under high-glucose conditions (25 mM D-glucose). Scale bar = 5 μm. H, J The number of red (autolysosome) and yellow puncta (autophagosome) per cell was determined using ImageJ. Twenty cells from six independent experiments were analyzed in each group. The data are shown as the mean ± SD (n = 6). *p < 0.05. **p < 0.01. 293T cells human embryonic kidney 293T, Co-IP coimmunoprecipitation, IP immunoprecipitation, K108E mutant Lys-to-Glu (K-to-E) substitution to mimic succinylation overexpression.