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. For in vivo validation, 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 N -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-31 in vitro and in vivo .