Slowing ribosome velocity restores folding and function of mutant CFTR
Slowing ribosome velocity restores folding and function of mutant CFTR
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DOI：
10.1172/jci124282
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发表时间：
2019-12-02
影响因子：
15.9
通讯作者：
Sorscher, Eric J.
中科院分区：
文献类型：
--
作者：
Oliver, Kathryn E.;Rauscher, Robert;Sorscher, Eric J.
囊性纤维化（CF）是由CF跨膜传导调节因子（CFTR）突变引起的，约90%的患者携带至少一个疾病相关变体F508 del拷贝。我们利用酵母表型系统来鉴定F508 del-CFTR生物发生的遗传修饰剂，其中核糖体蛋白L12（RPL 12/uL 11）作为分子靶标出现。在本研究中，我们研究了抑制RPL 12拯救F508 del蛋白合成和活性的机制。使用核糖体分析，我们发现翻译起始和延伸的速率被RPL 12沉默显著减慢。然而，蛋白水解稳定性和膜片钳测定显示RPL 12耗竭显著增加F508 del-CFTR稳态表达、结构域间组装和基线开放通道概率。我们接下来评估了Rpl 12校正的F508 del-CFTR是否可以通过伴随的药理学修复（例如，使用临床上批准的调节剂Lumacaftor和Tezacaftor），并证明了这些治疗的加和性。Rpl 12敲除还部分恢复了除F508 del之外的特异性CFTR变体的成熟，并且WT Cftr生物合成在Rpl 12单倍型充足小鼠的胰腺、结肠和回肠中增强。因此，核糖体速度的调节代表了理解CF发病机制和治疗反应的稳健方法。
Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), with approximately 90% of patients harboring at least one copy of the disease-associated variant F508del. We utilized a yeast phenomic system to identify genetic modifiers of F508del-CFTR biogenesis, from which ribosomal protein L12 (RPL12/uL11) emerged as a molecular target. In the present study, we investigated mechanism(s) by which suppression of RPL12 rescues F508del protein synthesis and activity. Using ribosome profiling, we found that rates of translation initiation and elongation were markedly slowed by RPL12 silencing. However, proteolytic stability and patch-clamp assays revealed RPL12 depletion significantly increased F508del-CFTR steady-state expression, interdomain assembly, and baseline open-channel probability. We next evaluated whether Rpl12-corrected F508del-CFTR could be further enhanced with concomitant pharmacologic repair (e.g., using clinically approved modulators lumacaftor and tezacaftor) and demonstrated additivity of these treatments. Rpl12 knockdown also partially restored maturation of specific CFTR variants in addition to F508del, and WT Cftr biogenesis was enhanced in the pancreas, colon, and ileum of Rpl12 haplosufficient mice. Modulation of ribosome velocity therefore represents a robust method for understanding both CF pathogenesis and therapeutic response.