Genome-Wide CRISPR Screen Identifies Phospholipid Scramblase 3 as the Biological Target of Mitoprotective Drug SS-31.

KEY POINTS: Szeto–Schiller-31–mediated mitoprotection is phospholipid scramblase 3–dependent. Phospholipid scramblase 3 is required for recovery after AKI. BACKGROUND: The synthetic tetrapeptide Szeto–Schiller (SS)-31 shows promise in alleviating mitochondrial dysfunction associated with common diseases. However, the precise pharmacological basis of its mitoprotective effects remains unknown. METHODS: To uncover the biological targets of SS-31, we performed a genome-scale clustered regularly interspaced short palindromic repeats screen in human kidney-2, a cell culture model where SS-31 mitigates cisplatin-associated cell death and mitochondrial dysfunction. The identified hit candidate gene was functionally validated using knockout cell lines, small interfering RNA-mediated downregulation, and tubular epithelial–specific conditional knockout mice. Biochemical interaction studies were also performed to examine the interaction of SS-31 with the identified target protein. RESULTS: Our primary screen and validation studies in hexokinase 2 and primary murine tubular epithelial cells showed that phospholipid scramblase 3 (PLSCR3), an understudied inner mitochondrial membrane protein, was essential for the protective effects of SS-31. Forvalidation, we generated tubular epithelial–specific knockout mice and found that Plscr3 gene ablation did not influence kidney function under normal conditions or affect the severity of cisplatin and rhabdomyolysis-associated AKI. However, Plscr3 gene deletion completely abrogated the protective effects of SS-31 during cisplatin and rhabdomyolysis-associated AKI. Biochemical studies showed that SS-31 directly binds to a previously uncharacterized-terminal domain and stimulates PLSCR3 scramblase activity. Finally, PLSCR3 protein expression was found to be increased in the kidneys of patients with AKI. CONCLUSIONS: PLSCR3 was identified as the essential biological target that facilitated the mitoprotective effects of SS-31and.