iProXProteomicsZhengquan YuHomo Sapienshttp://www.iprox.org/page/project.html?id=IPX0008342000zyu@cau.edu.cnCenter for Life ScienceiProXAberrant release of mitochondrial reactive oxygen species (mtROS) in response to cellular stress is well known for promoting cancer progression. However, precise molecular mechanism by which mtROS contribute to epithelial cancer progression remains only partially understood. Here, using colorectal cancer (CRC) models, we show that upon sensing excessive mtROS, phosphatase PGAM5, which normally localizes to the mitochondria, undergoes aberrant cleavage by presenilin-associated rhomboid-like protein (PARL), becoming released into the cytoplasm. Cytosolic PGAM5 then directly binds to and dephosphorylates MST3 kinase. This, in turn, prevents STK25-mediated LATS1/2 phosphorylation, leading to YAP activation and CRC progression. Importantly, depletion of MST3 reciprocally promotes accumulation of cytosolic PGAM5 by inducing mitochondrial damage. Taken together, these findings demonstrate how mtROS promotes CRC progression by activating YAP via a post-transcriptional positive feedback loop between PGAM5 and MST3, both of which can serve as potential targets for developing next-generation anti-colon cancer therapeutics.ROS-induced cytosolic release of mitochondrial PGAM5 promotes colorectal cancer progression by interacting with MST3.Wang Shiyang S, Wu Xi X, Bi Wenxin W, Xu Jiuzhi J, Hou Liyuan L, Li Guilin G, Pan Yuwei Y, Zhang Hanfu H, Li Mengzhen M, Du Sujuan S, Zhang Mingxin M, Liu Di D, Jin Shuiling S, Shi Xiaojing X, Tian Yuhua Y, Shuai Jianwei J, Plikus Maksim V MV, Song Moshi M, Zhou Zhaocai Z, Yu Lu L, Lv Cong C, Yu Zhengquan ZfalseMass spectrometry of HCT116 cellsHCT116 cells were transfected with flag-pcDNA3.1 plasmids or flag-MST3 plasmids. Whole-cell extracts were prepared with a lysis buffer, lysed on ice for 10 min, centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was collected. The anti-Flag antibody and protein A/G beads were added and incubated for 6-8 h and washed five times with wash buffer. Proteins were eluted with 0.15 M glycine (pH 2.5-3.1), and the supernatant was collected and immediately added to 1/10 volume of neutralization buffer (0.1 M NaOH) to adjust the pH of the eluted product to neutral. Mass spectrometry analysis was performed.Sat Mar 09 00:00:00 GMT 2024PXD050513960639915446