Kahali Smitaroopa, Baisya Ranojoy, Das Sayani, Datta Ankona
Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai 400005, India.
JACS Au. 2025 Jan 15;5(1):343-352. doi: 10.1021/jacsau.4c01021. eCollection 2025 Jan 27.
Hydrogen peroxide (HO) plays a critical role in the regulation and progress of autophagy, an essential recycling process that influences cellular homeostasis and stress response. Autophagy is characterized by the formation of intracellular vesicles analogous to recycle "bags" called autophagosomes, which fuse with lysosomes to form autolysosomes, eventually ending up as lysosomes. We have developed two novel autophagic vesicle-targeted peptide-based sensors, for HO and for pH, to simultaneously track HO and pH dynamics within autophagic vesicles as autophagy advances. Since pH values progressively decrease within autophagic vesicles with the progress of autophagy, we utilized information on vesicular pH to identify stages of autophagic vesicles in live cells. Fluorescence intensities of the HO sensor, , within autophagic compartments at different autophagic stages, which were identified by simultaneous pH mapping, revealed that HO levels vary significantly within autophagic vesicles as autophagy progresses, with maximum HO levels in the autolysosomal stage. This study provides the first detailed observation of HO fluctuations within autophagic vesicles throughout the entire process of autophagy in living mammalian cells, offering insights into the oxidative changes associated with this vital cellular process.
过氧化氢（HO）在自噬的调节和进程中发挥着关键作用，自噬是一种重要的循环过程，影响细胞内稳态和应激反应。自噬的特征是形成类似于回收“袋”的细胞内囊泡，称为自噬体，自噬体与溶酶体融合形成自噬溶酶体，最终以溶酶体告终。我们开发了两种新型的基于自噬囊泡靶向肽的传感器，一种用于检测HO，另一种用于检测pH，以便在自噬进行时同时追踪自噬囊泡内的HO和pH动态变化。由于随着自噬的进行，自噬囊泡内的pH值会逐渐降低，我们利用囊泡pH值的信息来识别活细胞中自噬囊泡的阶段。通过同时进行pH映射确定的不同自噬阶段的自噬隔室内，HO传感器的荧光强度表明，随着自噬的进行，自噬囊泡内的HO水平有显著变化，在自噬溶酶体阶段HO水平最高。这项研究首次详细观察了活的哺乳动物细胞自噬全过程中自噬囊泡内HO的波动情况，为了解与这一重要细胞过程相关的氧化变化提供了见解。
