Division of Pharmaceutics and Pharmacology, College of Pharmacy and Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio.
Department of Genetics, Genomics, and Informatics; College of Medicine, The University of Tennessee Health Science Center, Memphis, Tennessee.
J Am Soc Nephrol. 2024 Jun 1;35(6):681-695. doi: 10.1681/ASN.0000000000000338. Epub 2024 Mar 26.
Szeto–Schiller-31–mediated mitoprotection is phospholipid scramblase 3–dependent. Phospholipid scramblase 3 is required for recovery after AKI.
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.
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.
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 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 -terminal domain and stimulates PLSCR3 scramblase activity. Finally, PLSCR3 protein expression was found to be increased in the kidneys of patients with AKI.
PLSCR3 was identified as the essential biological target that facilitated the mitoprotective effects of SS-31 and .
Szeto-Schiller-31 介导的线粒体保护作用依赖于磷脂翻转酶 3。磷脂翻转酶 3 是急性肾损伤后恢复所必需的。
合成的四肽 Szeto-Schiller（SS）-31 在缓解与常见疾病相关的线粒体功能障碍方面显示出前景。然而，其确切的线粒体保护作用的药理学基础仍不清楚。
为了揭示 SS-31 的生物学靶点，我们在人肾-2 细胞培养模型中进行了全基因组规模的成簇规律间隔短回文重复（CRISPR）筛选，在该模型中，SS-31 减轻顺铂相关的细胞死亡和线粒体功能障碍。使用敲除细胞系、小干扰 RNA 介导的下调以及肾小管上皮细胞特异性条件性敲除小鼠对鉴定出的候选基因进行功能验证。还进行了生化相互作用研究，以检查 SS-31 与鉴定出的靶蛋白的相互作用。
我们在己糖激酶 2 和原代鼠肾小管上皮细胞中的初步筛选和验证研究表明，磷脂翻转酶 3（PLSCR3）是一种研究较少的线粒体内膜蛋白，对于 SS-31 的保护作用至关重要。为了验证，我们生成了肾小管上皮细胞特异性敲除小鼠，并发现 Plscr3 基因缺失在正常条件下不影响肾功能，也不影响顺铂和横纹肌溶解相关急性肾损伤的严重程度。然而，Plscr3 基因缺失完全消除了 SS-31 在顺铂和横纹肌溶解相关急性肾损伤中的保护作用。生化研究表明，SS-31 直接与以前未表征的 C 端结构域结合并刺激 PLSCR3 翻转酶活性。最后，在急性肾损伤患者的肾脏中发现 PLSCR3 蛋白表达增加。
PLSCR3 被鉴定为必需的生物学靶点，促进了 SS-31 的线粒体保护作用，并可能作为急性肾损伤的治疗靶点。
