Karapurkar Janardhan Keshav, Rajkumar Sripriya, Jung Ji-Hye, Kim Ji-Young, Birappa Girish, Gowda D A Ayush, Colaco Jencia Carminha, Suresh Bharathi, Choi Jung-Yun, Woo Sang Hyeon, Jo Won-Jun, Lee Jong-Hee, Kim Kye-Seong, Hong Seok-Ho, Ramakrishna Suresh
Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, 04763, South Korea.
Department of Internal Medicine, College of Medicine, Kangwon National University, Chuncheon, South Korea.
Theranostics. 2025 Mar 19;15(10):4526-4549. doi: 10.7150/thno.105994. eCollection 2025.
Interstitial lung disease (ILD) is a pulmonary disorder characterized by a combination of inflammation and fibrosis in the lung parenchyma, which initiates with the dysfunction of alveolar epithelial cells (AECs). The alveolar cells secrete surfactant proteins that lowers the surface tension of fluids in the lungs and maintains the stability of pulmonary tissue. Mutations on surfactant protein C (SFTPC), particularly I73T, are associated with a toxic gain of function that causes misfolding and the accumulation of immature SFTPC proteins, triggering pulmonary fibrosis (PF). Therefore, it is crucial to block the accumulation of the SFTPC protein during ILD progression. We used a loss-of-function-based CRISPR/Cas9 library kit to screen genome-wide for deubiquitinating enzymes that regulate the SFTPC protein. The interaction between USP11 and SFTPC and its ubiquitination status was validated by immunoprecipitation and the TUBEs assay. HDR-directed knock-in of the I73T mutation into the locus in human induced pluripotent stem cells (hiPSCs) was performed using the CRISPR/Cas9 system, and then those cells were differentiated into alveolar organoids (AOs) using a forced aggregation protocol. The clinical relevance of the USP11 inhibitor and its effect on preventing PF were investigated in a TGF-β-induced fibrosis in AOs and bleomycin (BLM)-induced mouse model. We identified USP11 as a novel deubiquitinase that interacts with, stabilizes, deubiquitinates, and extends the half-life of SFTPC. Remarkably, USP11 stabilized and prolonged the half-life of the SFTPC mutant protein significantly more than the wild type. In vitro functional studies revealed that USP11 exacerbates SFTPC -induced fibrosis and enhances the epithelial-to-mesenchymal transition. Furthermore, we present a human in vitro model for investigating -induced fibrosis: hiPSCs-derived-AOs carrying the pathogenic variant. Interestingly, USP11 depletion in the organoids mitigated SFTPC -induced fibrosis. Finally, pharmacological inhibition of USP11 prevented PF caused by TGF-β in hiPSCs-SFTPC-AOs and BLM-induced mouse model, underscoring its therapeutic potential. Altogether, USP11 is a major protein stabilizer of SFTPC, and the clinical inhibition of USP11 during PF could be a novel therapeutic approach for ILD patients.
间质性肺疾病（ILD）是一种肺部疾病，其特征是肺实质出现炎症和纤维化，始于肺泡上皮细胞（AEC）功能障碍。肺泡细胞分泌表面活性蛋白，可降低肺内液体的表面张力并维持肺组织的稳定性。表面活性蛋白C（SFTPC）的突变，尤其是I73T，与功能获得性毒性相关，导致未成熟SFTPC蛋白错误折叠和积累，引发肺纤维化（PF）。因此，在ILD进展过程中阻断SFTPC蛋白的积累至关重要。我们使用基于功能缺失的CRISPR/Cas9文库试剂盒在全基因组范围内筛选调节SFTPC蛋白的去泛素化酶。通过免疫沉淀和TUBEs分析验证了USP11与SFTPC之间的相互作用及其泛素化状态。使用CRISPR/Cas9系统将I73T突变通过同源定向修复敲入人诱导多能干细胞（hiPSC）的基因座，然后使用强制聚集方案将这些细胞分化为肺泡类器官（AO）。在AO中由转化生长因子-β（TGF-β）诱导的纤维化和博来霉素（BLM）诱导的小鼠模型中研究了USP11抑制剂的临床相关性及其对预防PF的作用。我们确定USP11是一种新型去泛素酶，它与SFTPC相互作用、使其稳定、去泛素化并延长其半衰期。值得注意的是，USP11使SFTPC突变蛋白的半衰期稳定并延长的程度明显超过野生型。体外功能研究表明，USP11会加剧SFTPC诱导的纤维化并增强上皮-间质转化。此外，我们提供了一种用于研究诱导纤维化的人源体外模型：携带致病性变体的hiPSC衍生的AO。有趣的是，类器官中USP11的缺失减轻了SFTPC诱导的纤维化。最后，对USP11的药理抑制在hiPSC-SFTPC-AO中预防了TGF-β引起PF，并在BLM诱导的小鼠模型中发挥作用，突出了其治疗潜力。总之，USP11是SFTPC的主要蛋白稳定剂，在PF期间对USP11进行临床抑制可能是ILD患者的一种新型治疗方法。
