Cyclin D-CDK4 Disulfide Bond Attenuates Pulmonary Vascular Cell Proliferation.
Cyclin D-CDK4 Disulfide Bond Attenuates Pulmonary Vascular Cell Proliferation.
复制标题
细胞周期蛋白D-CDK4二硫键抑制肺血管细胞增殖。
DOI：
10.1161/circresaha.122.321836
复制
发表时间：
2023-12-08
影响因子：
20.1
通讯作者：
Rudyk, Olena
中科院分区：
文献类型：
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作者：
Knight, Hannah;Abis, Giancarlo;Kaur, Manpreet;Green, Hannah L. H.;Krasemann, Susanne;Hartmann, Kristin;Lynham, Steven;Clark, James;Zhao, Lan;Ruppert, Clemens;Weiss, Astrid;Schermuly, Ralph T.;Eaton, Philip;Rudyk, Olena
肺动脉高压（PH）是一种慢性血管疾病，其特征是平滑肌细胞过度增殖和细胞氧化还原和代谢平衡紊乱。氧化剂诱导细胞周期停滞停止增殖，但是，很少有人知道的氧化还原调节效应蛋白介导这些过程。在这里，我们报告了一种新的激酶抑制二硫键的细胞周期蛋白D-CDK 4（细胞周期蛋白依赖性激酶4），并探讨其在细胞增殖和PH的作用。氧化修饰的细胞周期蛋白D-CDK 4检测在人肺动脉平滑肌细胞和人肺动脉内皮细胞。利用定点诱变、串联质谱、基于细胞的实验、体外激酶活性测定、计算机结构建模和新型氧化还原死亡组成型敲入小鼠来研究CDK 4半胱氨酸修饰在肺血管细胞增殖中的性质并明确确立其重要性。此外，在肺动脉高压患者的肺动脉和分离的人肺动脉平滑肌细胞中以及在PH的3种临床前模型中，在体内评估了细胞周期蛋白D-CDK 4氧化。细胞周期蛋白D-CDK 4分别在C7/8和C135之间形成可逆的氧化剂诱导的异源二聚体二硫键二聚体，在体外细胞和体内肺动脉中抑制细胞周期蛋白D-CDK 4激酶活性，降低Rb（视网膜母细胞瘤）蛋白磷酸化，并诱导细胞周期停滞。CDK 4 C135突变导致激酶受损表型，其降低了实验小鼠PH模型中的细胞增殖率并加重了疾病表型，表明这种半胱氨酸对于细胞周期蛋白D-CDK 4激酶活性是不可或缺的。来自肺动脉高压患者的肺动脉和人肺动脉平滑肌细胞显示CDK 4二硫化物水平降低，与CDK 4在人肺动脉高压中过度活跃一致。此外，诱导细胞周期蛋白D-CDK 4二硫化物的金诺芬治疗通过减轻肺血管重塑来减轻实验性PH模型中的疾病严重程度。细胞周期蛋白D-CDK 4中的一个新的二硫键作为一个快速开关抑制激酶活性和停止细胞增殖。这种氧化修饰在关键的半胱氨酸残基处形成，这是CDK 4所特有的，为设计预测在PH中有益的选择性共价抑制剂提供了可能性。
Pulmonary hypertension (PH) is a chronic vascular disease characterized, among other abnormalities, by hyperproliferative smooth muscle cells and a perturbed cellular redox and metabolic balance. Oxidants induce cell cycle arrest to halt proliferation; however, little is known about the redox-regulated effector proteins that mediate these processes. Here, we report a novel kinase-inhibitory disulfide bond in cyclin D-CDK4 (cyclin-dependent kinase 4) and investigate its role in cell proliferation and PH. Oxidative modifications of cyclin D-CDK4 were detected in human pulmonary arterial smooth muscle cells and human pulmonary arterial endothelial cells. Site-directed mutagenesis, tandem mass-spectrometry, cell-based experiments, in vitro kinase activity assays, in silico structural modeling, and a novel redox-dead constitutive knock-in mouse were utilized to investigate the nature and definitively establish the importance of CDK4 cysteine modification in pulmonary vascular cell proliferation. Furthermore, the cyclin D-CDK4 oxidation was assessed in vivo in the pulmonary arteries and isolated human pulmonary arterial smooth muscle cells of patients with pulmonary arterial hypertension and in 3 preclinical models of PH. Cyclin D-CDK4 forms a reversible oxidant-induced heterodimeric disulfide dimer between C7/8 and C135, respectively, in cells in vitro and in pulmonary arteries in vivo to inhibit cyclin D-CDK4 kinase activity, decrease Rb (retinoblastoma) protein phosphorylation, and induce cell cycle arrest. Mutation of CDK4 C135 causes a kinase-impaired phenotype, which decreases cell proliferation rate and alleviates disease phenotype in an experimental mouse PH model, suggesting this cysteine is indispensable for cyclin D-CDK4 kinase activity. Pulmonary arteries and human pulmonary arterial smooth muscle cells from patients with pulmonary arterial hypertension display a decreased level of CDK4 disulfide, consistent with CDK4 being hyperactive in human pulmonary arterial hypertension. Furthermore, auranofin treatment, which induces the cyclin D-CDK4 disulfide, attenuates disease severity in experimental PH models by mitigating pulmonary vascular remodeling. A novel disulfide bond in cyclin D-CDK4 acts as a rapid switch to inhibit kinase activity and halt cell proliferation. This oxidative modification forms at a critical cysteine residue, which is unique to CDK4, offering the potential for the design of a selective covalent inhibitor predicted to be beneficial in PH.