去泛素化酵素USP45藉由調節肌動蛋白之動態性以調控細胞自噬及溶酶體的活性

Abstract

蛋白質恆定對細胞健康至關重要，是由泛素-蛋白酶體系統（UPS）和細胞自噬維持。自噬是一種具演化保留性的過程，藉由與溶酶體融合，降解包覆其中的不正常折疊蛋白或受損胞器。近年來，越來越多的研究顯示，衰老與自噬、溶酶體功能下降相關。然而，詳細的機制和有效的療法仍然不明。本研究發現去泛素化酵素dUSP45/USP45的新功能，它在果蠅和哺乳動物系統中調節自噬和溶酶體活性。我們的發現USP45使得溶酶體上的液胞型ATP酶水平下降，從而降低了溶酶體的酸化程度。與過去關於USP45潛在相互作用蛋白的質譜分析報告一致，我們的免疫沉澱結果顯示，Coronin1B（Coro1B）與USP45相互作用。此外，USP45介導了Coro1B的去泛素化，並維持其蛋白穩定性。Coro1B已知是肌動蛋白之調節蛋白，因此我們在USP45剔除的細胞中發現了共定位的溶酶體與肌動蛋白斑塊結構。進一步我們的數據也顯示，肌動蛋白聚合抑制劑Latrunculin A（LatA）和CK666會破壞液胞型ATP酶和溶酶體的共定位，進一步支持肌動蛋白在自噬調控中的作用。而Latrunculin A（LatA）沖刷實驗證明，通過置換正常培養基，可以顯著恢復USP45敲除細胞中的溶酶體活性以及肌動蛋白和溶酶體的共定位。有趣的是，我們發現，USP45敲除所導致的液胞型ATP酶水平升高取決於WASP，而不是WASH。如同我們的推測，Coro1B在抑制細胞自噬和溶酶體上的液胞型ATP酶含量，發揮了與USP45類似的作用。為了評估USP45的生理功能，我們進行了RT-PCR和西方墨點分析，顯示dUSP45 mRNA水平與成熟的Cathepsin蛋白含量在衰老過程中呈現負相關。此外，通過敲除dUSP45，可以顯著恢復HTT-多谷氨酰胺（polyQ）所引發的退化。我們的研究揭示了USP45在藉由肌動蛋白對自噬及溶酶體進行調節的新功能，以及其對溶酶體液胞型ATP酶水平的影響，並暗示USP45可能成為與衰老相關疾病的潛在治療目標。

Proteostasis is beneficial to cellular health, which is maintained by the ubiquitin-proteasome system (UPS) and autophagy. As an evolutionary conserved process, autophagy degrades engulfed material including misfolded protein or damaged organelles through fusion with lysosome. Recent years, increasing studies describing that aging is associated with function decline of autophagy/lysosome. However, the detail mechanism and efficient therapy remain to be discovered. This study identifies the novel function of deubiquitinase, dUSP45/USP45, which modulates autophagy and lysosomal activity in Drosophila and mammalian systems. Our findings reveal that USP45 attenuates V-ATPase level on lysosome, leading to reduced lysosomal acidification. Consistent with previous report which characterized potential interacting protein of USP45 in MS analysis, our immunoprecipitation results show Coronin1B (Coro1B) interacts with USP45. Additionally, USP45 mediates deubiquitination of Coro1B and maintains its protein stability. Coro1B is regarded as actin dynamics regulator protein, we thus find the actin patch-like structure colocalized with lysosome in USP45-depleted cell. Moreover, our data show that colocalization of V-ATPase and lysosome is disrupted by actin polymerization inhibitors, Latrunculin A (LatA) and CK666, further supporting the role of actin dynamics in autophagy regulation. Furthermore, Latrunculin A (LatA) washout experiments demonstrate that lysosomal activity and actin-lysosome colocalization are dramatically restored by replenishment of normal medium in USP45 knockdown cells. Intriguingly, we find that USP45 knockdown-induced elevation of V-ATPase level on lysosome is dependent on WASP, rather than WASH. As our speculation, Coro1B plays a similar role of USP45 in inhibiting autophagy flux and V-ATPase level on lysosome. To evaluate the physiological function of USP45, we conduct RT-PCR and western blot analyses, revealing a negative correlation between dUSP45 mRNA levels and mature cathepsin protein levels during aging. Furthermore, HTT-polygluatmine (polyQ)-induced degeneration are significantly recovered by knockdown dUSP45. Our study unravels the novel function of USP45 in autophagy/lysosome modulation through actin dynamics and its impact on lysosomal V-ATPase level and suggests USP45 as potential therapeutic target for age-related diseases.