Robert Wood Johnson Medical School , Rutgers University , New Brunswick , New Jersey , USA.
Center for Advanced Biotechnology & Medicine , Rutgers University , Piscataway , New Jersey , USA.
Hepatology. 2023 Jan 1;77(1):144-158. doi: 10.1002/hep.32574. Epub 2022 Jun 13.
Hepatocyte keratin polypeptides 8/18 (K8/K18) are unique among intermediate filaments proteins (IFs) in that their mutation predisposes to, rather than causes, human disease. Mice that overexpress human K18 R90C manifest disrupted hepatocyte keratin filaments with hyperphosphorylated keratins and predisposition to Fas-induced liver injury. We hypothesized that high-throughput screening will identify compounds that protect the liver from mutation-triggered predisposition to injury.
Using A549 cells transduced with a lentivirus K18 construct and high-throughput screening, we identified the SRC-family tyrosine kinases inhibitor, PP2, as a compound that reverses keratin filament disruption and protects from apoptotic cell death caused by K18 R90C mutation at this highly conserved arginine. PP2 also ameliorated Fas-induced apoptosis and liver injury in male but not female K18 R90C mice. The PP2 male selectivity is due to its lower turnover in male versus female livers. Knockdown of SRC but not another kinase target of PP2, protein tyrosine kinase 6, in A549 cells abrogated the hepatoprotective effect of PP2. Phosphoproteomic analysis and validation showed that the protective effect of PP2 associates with Ser/Thr but not Tyr keratin hypophosphorylation, and differs from the sex-independent effect of the Ser/Thr kinase inhibitor PKC412. Inhibition of RAF kinase, a downstream target of SRC, by vemurafenib had a similar protective effect to PP2 in A549 cells and male K18 R90C mice.
PP2 protects, in a male-selective manner, keratin mutation-induced mouse liver injury by inhibiting SRC-triggered downstream Ser/Thr phosphorylation of K8/K18, which is phenocopied by RAF kinase inhibitor vemurafenib. The PP2/vemurafenib-associated findings, and their unique mechanisms of action, further support the potential role of select kinase inhibition as therapeutic opportunities for keratin and other IF-associated human diseases.
肝细胞角蛋白 8/18（K8/K18）在中间丝蛋白（IFs）中是独一无二的，因为其突变易导致而非引起人类疾病。过表达人 K18 R90C 的小鼠表现出肝细胞角蛋白丝的破坏，伴有角蛋白的过度磷酸化和 Fas 诱导的肝损伤易感性。我们假设高通量筛选将鉴定出保护肝脏免受突变引发的损伤易感性的化合物。
我们使用携带 K18 构建体的慢病毒转导的 A549 细胞进行高通量筛选，鉴定出 SRC 家族酪氨酸激酶抑制剂 PP2 是一种可逆转角蛋白丝破坏并防止 K18 R90C 突变引起的细胞凋亡的化合物。在该高度保守的精氨酸处。PP2 还改善了 Fas 诱导的雄性而非雌性 K18 R90C 小鼠的凋亡和肝损伤。PP2 的雄性选择性是由于其在雄性肝脏中的周转率低于雌性肝脏。在 A549 细胞中敲低 SRC 而不是 PP2 的另一个激酶靶标蛋白酪氨酸激酶 6，可消除 PP2 的肝保护作用。磷酸化蛋白质组学分析和验证表明，PP2 的保护作用与丝氨酸/苏氨酸而非酪氨酸角蛋白低磷酸化相关，并且与 Ser/Thr 激酶抑制剂 PKC412 的性别无关作用不同。SRC 的下游靶标 RAF 激酶抑制剂 vemurafenib 在 A549 细胞和雄性 K18 R90C 小鼠中具有与 PP2 相似的保护作用。
PP2 以雄性选择性的方式通过抑制 SRC 触发的 K8/K18 的 Ser/Thr 磷酸化来保护角蛋白突变诱导的小鼠肝损伤，这被 RAF 激酶抑制剂 vemurafenib 模拟。PP2/vemurafenib 相关发现及其独特的作用机制进一步支持选择性激酶抑制作为角蛋白和其他 IF 相关人类疾病的治疗机会的潜力。
