| Summary: | 博士 === 國立陽明大學 === 生命科學系暨基因體科學研究所 === 106 === Aging and age-related diseases are important problems in the “super-aged” societies. Therefore, investigation of the molecular mechanism could provide valuable knowledge about how to slow down and even reverse the aging process. Although Cisd2 has been identified as a key regulator of mammalian lifespan, the detailed mechanism is largely unknown. Importantly, one critical phenotype shared in Cisd2 KO and natural aging is the degenerative loss of skeletal muscle. Age-related muscle loss, especially in fast-twitch fibers, not only accounts for frailty but also contribute to the systemic metabolic dysregulation. The aim of this study is to investigate the functional network of Cisd2, an age-associated protein, to discover mechanisms how Cisd2 deficiency causes muscle atrophy and muscle aging. A label-free quantitative proteomic investigation of the gastrocnemius of naturally aged, systemic Cisd2 KO, muscle-specific Cisd2 knockout (mKO), and Cisd2 TG mice were performed. With proteomic pathway analysis, calcium signaling and unfolded protein response (UPRER) were the top two significant pathways during natural aging and Cisd2 deficiency. Importantly, Serca1 pump activity was significantly compromised in both the natural aging and Cisd2 mKO mice. This functional impairment could be attributable to the cysteine sulfonation and tyrosine nitration of Serca1 protein. Taken together, most of the muscle phenotypes observed in Cisd2 mKO mice were also detectable in the naturally aged mice in terms of pathological alterations, ultrastructural abnormalities, Serca1 impairment, and UPR. Furthermore, GST pull down experiments were used to identify the Cisd2-interacing molecules mediating downstream pathways. The interactome map of diverse tissues was generated, and the pulled down proteins were subjected to mass spectrometric analysis for identification of Cisd2-interacting proteins in liver and gastrocnemius. Among them, two putative interacting partners, Casq1 and Serca1, were further characterized for validation. In addition, the potential phosphorylation site of Cisd2 has also been identified by LC-MS/MS, albeit the statistical significance was not very high. Whether the T106 phosphorylation affects Cisd2 cluster transfer activity was also tested using phosphor-mimic Cisd2 T106D and apo-CIAPIN1 in vitro. Taken together, this work substantiates the hypothesis that Cisd2 is crucial to the gastrocnemius muscle and suggests that Cisd2 is a potential therapeutic target for muscle aging. Knowledge about the Cisd2 activity, post-translational modification, and interacting proteins would improve our understanding of this novel age-related Cisd2 protein.
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