Shi Bo, Huang Qi-Quan, Birkett Robert, Doyle Renee, Dorfleutner Andrea, Stehlik Christian, He Congcong, Pope Richard M
a Division of Rheumatology , Feinberg School of Medicine, Northwestern University , Chicago , IL , USA.
b Department of Cell and Molecular Biology , Feinberg School of Medicine, Northwestern University , Chicago , IL , USA.
Autophagy. 2017 Feb;13(2):285-301. doi: 10.1080/15548627.2016.1261238. Epub 2016 Dec 8.
We previously observed that SNAPIN, which is an adaptor protein in the SNARE core complex, was highly expressed in rheumatoid arthritis synovial tissue macrophages, but its role in macrophages and autoimmunity is unknown. To identify SNAPIN's role in these cells, we employed siRNA to silence the expression of SNAPIN in primary human macrophages. Silencing SNAPIN resulted in swollen lysosomes with impaired CTSD (cathepsin D) activation, although total CTSD was not reduced. Neither endosome cargo delivery nor lysosomal fusion with endosomes or autophagosomes was inhibited following the forced silencing of SNAPIN. The acidification of lysosomes and accumulation of autolysosomes in SNAPIN-silenced cells was inhibited, resulting in incomplete lysosomal hydrolysis and impaired macroautophagy/autophagy flux. Mechanistic studies employing ratiometric color fluorescence on living cells demonstrated that the reduction of SNAPIN resulted in a modest reduction of H pump activity; however, the more critical mechanism was a lysosomal proton leak. Overall, our results demonstrate that SNAPIN is critical in the maintenance of healthy lysosomes and autophagy through its role in lysosome acidification and autophagosome maturation in macrophages largely through preventing proton leak. These observations suggest an important role for SNAPIN and autophagy in the homeostasis of macrophages, particularly long-lived tissue resident macrophages.
我们之前观察到，作为SNARE核心复合体中的衔接蛋白，SNAPIN在类风湿性关节炎滑膜组织巨噬细胞中高表达，但其在巨噬细胞和自身免疫中的作用尚不清楚。为了确定SNAPIN在这些细胞中的作用，我们使用小干扰RNA（siRNA）沉默原代人巨噬细胞中SNAPIN的表达。沉默SNAPIN导致溶酶体肿胀，组织蛋白酶D（CTSD）激活受损，尽管CTSD总量未减少。在强制沉默SNAPIN后，内体货物运输以及溶酶体与内体或自噬体的融合均未受到抑制。SNAPIN沉默细胞中溶酶体的酸化和自噬溶酶体的积累受到抑制，导致溶酶体水解不完全和巨自噬/自噬通量受损。利用活细胞上的比率色荧光进行的机制研究表明，SNAPIN的减少导致H⁺泵活性适度降低；然而，更关键的机制是溶酶体质子泄漏。总体而言，我们的结果表明，SNAPIN在维持健康的溶酶体和自噬方面至关重要，其在巨噬细胞的溶酶体酸化和自噬体成熟中发挥作用，主要是通过防止质子泄漏。这些观察结果表明SNAPIN和自噬在巨噬细胞的稳态中具有重要作用，尤其是在长寿的组织驻留巨噬细胞中。
