Background:PALMD (palmdelphin) belongs to the family of paralemmin proteins implicated in cytoskeletal regulation. Single nucleotide polymorphisms in the PALMD locus that result in reduced expression are strong risk factors for development of calcific aortic valve stenosis and predict severity of the disease.Methods:Immunodetection and public database screening showed dominant expression of PALMD in endothelial cells (ECs) in brain and cardiovascular tissues including aortic valves. Mass spectrometry, coimmunoprecipitation, and immunofluorescent staining allowed identification of PALMD partners. The consequence of loss of PALMD expression was assessed in small interferring RNA-treated EC cultures, knockout mice, and human valve samples. RNA sequencing of ECs and transcript arrays on valve samples from an aortic valve study cohort including patients with the single nucleotide polymorphism rs7543130 informed about gene regulatory changes.Results:ECs express the cytosolic PALMD-KKVI splice variant, which associated with RANGAP1 (RAN GTP hydrolyase activating protein 1). RANGAP1 regulates the activity of the GTPase RAN and thereby nucleocytoplasmic shuttling via XPO1 (Exportin1). Reduced PALMD expression resulted in subcellular relocalization of RANGAP1 and XPO1, and nuclear arrest of the XPO1 cargoes p53 and p21. This indicates an important role for PALMD in nucleocytoplasmic transport and consequently in gene regulation because of the effect on localization of transcriptional regulators. Changes in EC responsiveness on loss of PALMD expression included failure to form a perinuclear actin cap when exposed to flow, indicating lack of protection against mechanical stress. Loss of the actin cap correlated with misalignment of the nuclear long axis relative to the cell body, observed in PALMD-deficient ECs, Palmd^−/− mouse aorta, and human aortic valve samples derived from patients with calcific aortic valve stenosis. In agreement with these changes in EC behavior, gene ontology analysis showed enrichment of nuclear- and cytoskeleton-related terms in PALMD-silenced ECs.Conclusions:We identify RANGAP1 as a PALMD partner in ECs. Disrupting the PALMD/RANGAP1 complex alters the subcellular localization of RANGAP1 and XPO1, and leads to nuclear arrest of the XPO1 cargoes p53 and p21, accompanied by gene regulatory changes and loss of actin-dependent nuclear resilience. Combined, these consequences of reduced PALMD expression provide a mechanistic underpinning for PALMD’s contribution to calcific aortic valve stenosis pathology.

中文翻译：

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Palmdelphin 调节核对内皮机械应力的弹性

背景：PALMD（palmdelphin）属于与细胞骨架调节有关的副脑蛋白家族。PALMD 中的单核苷酸多态性导致表达减少的基因座是发生钙化性主动脉瓣狭窄和预测疾病严重程度的重要危险因素。 . 质谱、免疫共沉淀和免疫荧光染色允许识别 PALMD 伙伴。在小干扰 RNA 处理的 EC 培养物、基因敲除小鼠和人类瓣膜样本中评估了 PALMD 表达丧失的后果。来自主动脉瓣研究队列的瓣膜样本的 ECs 和转录物阵列的 RNA 测序，包括具有单核苷酸多态性 rs7543130 的患者，了解基因调控变化。结果：ECs 表达细胞质PALMD-KKVI剪接变体，与 RANGAP1（RAN GTP 水解酶激活蛋白 1）相关。RANGAP1 调节 GTPase RAN 的活性，从而通过 XPO1 (Exportin1) 调节核质穿梭。PALMD 表达降低导致 RANGAP1 和 XPO1 的亚细胞重定位，以及 XPO1 货物 p53 和 p21 的核停滞。这表明 PALMD 在核细胞质运输中的重要作用，并因此在基因调控中发挥重要作用，因为它对转录调节因子的定位有影响。失去PALMD 时EC 反应性的变化表达包括在暴露于流动时未能形成核周肌动蛋白帽，表明缺乏对机械应力的保护。肌动蛋白帽的丢失与核长轴相对于细胞体的错位相关，在PALMD 缺陷的 EC、Palmd ^-/-小鼠主动脉和来自钙化性主动脉瓣狭窄患者的人主动脉瓣样本中观察到。与 EC 行为的这些变化一致，基因本体分析显示PALMD中核和细胞骨架相关术语的丰富-沉默的 ECs。结论：我们将 RANGAP1 确定为 ECs 中的 PALMD 伙伴。破坏 PALMD/RANGAP1 复合物会改变 RANGAP1 和 XPO1 的亚细胞定位，并导致 XPO1 货物 p53 和 p21 的核停滞，伴随着基因调控变化和肌动蛋白依赖性核弹性的丧失。结合起来，PALMD表达降低的这些后果为 PALMD 对钙化主动脉瓣狭窄病理的贡献提供了机制基础。