A single mutation in NFU1 gene metabolically reprograms pulmonary artery smooth muscle cells

NFU1 is an iron-sulfur (Fe-S) scaffold protein, involved in Fe-S assembly and transfer to a range of mitochondrial metalloproteins. Patients with the NFU1G208C mutation develop pulmonary arterial hypertension (PAH) with 70% penetrance. Rats with NFU1G206C homozygous mutation demonstrated the PAH phenotype showing increased pulmonary vasculature remodeling, right ventricular (RV) hypertrophy, and RV pressure. In the present study, we discovered phenotypic metabolic changes in pulmonary arterial smooth muscle cells (PASMC) from NFU1G206C homozygous mutant rats associated with alterations in the mitochondrial proteome. Quantitative analysis of the mitochondrial proteome showed significant changes in the abundance of 208 proteins involved in various metabolic and antioxidant functions in response to the NFU1 mutation. Our data indicates that the NFU1G206C homozygous mutant rats have decreased expression of complex I and II, which are known to depend on iron-sulfur clusters, and increased expression of complexes III to V, accompanied with a significant decrease in mitochondrial function, pyruvate dehydrogenase (PDH) activity and amplified glycolysis and anabolism in PASMC. The NFU1 mutation produced a dysregulated antioxidant system in the mitochondria which leads to increased levels of reactive oxygen species. Due to alterations in apoptosis regulating proteins, the NFU1G206C cells were found to exhibit high proliferation rates and resistance to apoptosis as compared with the wild type (WT). Finally, the NFU1G206C mitochondrial proteome showed significant abundance changes in proteins that regulate fatty acid (FA) metabolism and exhibited increased FA oxidation compared to WT, suggesting increased FA oxidation compensates for the decreased PDH activity. In conclusion, our data indicates that the NFU1G206C mutation induces a metabolic shift in the PASMC, which results in a hyper-proliferative and apoptosis resistant phenotype and presents a novel cellular model to study PAH.

NFU1是一种铁硫（Fe-S）支架蛋白，参与铁硫簇的组装并向多种线粒体金属蛋白转移。携带NFU1G208C突变的患者会患肺动脉高压（PAH），外显率达70%。带有NFU1G206C纯合突变的大鼠表现出PAH表型，包括肺血管重构加重、右心室（RV）肥厚及右心室压力升高。本研究中，我们发现来自NFU1G206C纯合突变大鼠的肺动脉平滑肌细胞（PASMC）存在表型性代谢改变，且该改变与线粒体蛋白质组的异常相关。对线粒体蛋白质组的定量分析显示，针对NFU1突变，共有208种参与多种代谢与抗氧化功能的蛋白质丰度发生显著变化。我们的数据表明，NFU1G206C纯合突变大鼠的复合物I和II的表达水平降低，这两种复合物已知依赖铁硫簇，而复合物III至V的表达水平升高，同时伴随线粒体功能、丙酮酸脱氢酶（PDH）活性显著下降，以及PASMC中糖酵解与合成代谢增强。NFU1突变导致线粒体抗氧化系统失调，进而使活性氧（reactive oxygen species, ROS）水平升高。由于凋亡调控蛋白的异常，与野生型（WT）细胞相比，NFU1G206C突变细胞表现出高增殖速率与凋亡抵抗性。最后，NFU1G206C的线粒体蛋白质组中，调控脂肪酸（FA）代谢的蛋白质丰度发生显著变化，且与野生型相比，其脂肪酸氧化水平升高，这提示脂肪酸氧化增强可代偿丙酮酸脱氢酶活性的下降。综上，我们的数据表明NFU1G206C突变诱导PASMC发生代谢重编程，进而产生高增殖与凋亡抵抗的表型，同时为肺动脉高压的研究提供了一种新型细胞模型。