Christopher R Brown, Madhur Shetty, James D Foster
{"title":"棕榈酰化调节去甲肾上腺素转运体的摄取、表面定位和总表达,对体位性正位性心动过速综合征具有致病影响。","authors":"Christopher R Brown, Madhur Shetty, James D Foster","doi":"10.1111/jnc.16241","DOIUrl":null,"url":null,"abstract":"<p><p>Postural orthostatic tachycardia syndrome (POTS) is an adrenergic signaling disorder characterized by excessive plasma norepinephrine, postural tachycardia, and syncope. The norepinephrine transporter (NET) modulates adrenergic homeostasis via the reuptake of extracellular catecholamines and is implicated in the pathogenesis of adrenergic and neurological disorders. In this study, we reveal NET is palmitoylated in male Sprague-Dawley rats and Lilly Laboratory Cell Porcine Kidney (LLC-PK<sub>1</sub>) cells. S-palmitoylation, or the addition of a 16-carbon saturated fatty acid, is a reversible post-translational modification responsible for the regulation of numerous biological mechanisms. We found that LLC-PK<sub>1</sub> NET is dynamically palmitoylated, and that inhibition with the palmitoyl acyltransferase (DHHC) inhibitor, 2-bromopalmitate (2BP) results in decreased NET palmitoylation within 90 min of treatment. This result was followed closely by a reduction in transport capacity, cell surface, and total cellular NET expression after 120 min of treatment. Increasing 2BP concentrations and treatment time revealed a nearly complete loss of total NET protein. Co-expression with individual DHHCs revealed a single DHHC enzyme, DHHC1, promoted wild-type (WT) hNET palmitoylation and elevated NET protein levels. The POTS-associated NET mutant, A457P, exhibits dramatically decreased transport capacity and cell surface levels which we have confirmed in the current study. In an attempt to recover A457P NET expression, we co-expressed the A457P variant with DHHC1 to drive expression as seen with the WT protein but instead saw an increase in NET N-terminal immuno-detectable forms and fragments. Elimination of a potential palmitoylation site at cysteine 44 in the N-terminal tail of hNET resulted in a low expression phenotype mimicking the A457P hNET variant. Further investigation of A457P NET palmitoylation and surface expression is necessary, but our preliminary novel findings reveal palmitoylation as a mechanism of NET regulation and suggest that dysregulation of this process may contribute to the pathogenesis of adrenergic disorders like POTS.</p>","PeriodicalId":16527,"journal":{"name":"Journal of Neurochemistry","volume":null,"pages":null},"PeriodicalIF":4.2000,"publicationDate":"2024-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Palmitoylation regulates norepinephrine transporter uptake, surface localization, and total expression with pathogenic implications in postural orthostatic tachycardia syndrome.\",\"authors\":\"Christopher R Brown, Madhur Shetty, James D Foster\",\"doi\":\"10.1111/jnc.16241\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Postural orthostatic tachycardia syndrome (POTS) is an adrenergic signaling disorder characterized by excessive plasma norepinephrine, postural tachycardia, and syncope. The norepinephrine transporter (NET) modulates adrenergic homeostasis via the reuptake of extracellular catecholamines and is implicated in the pathogenesis of adrenergic and neurological disorders. In this study, we reveal NET is palmitoylated in male Sprague-Dawley rats and Lilly Laboratory Cell Porcine Kidney (LLC-PK<sub>1</sub>) cells. S-palmitoylation, or the addition of a 16-carbon saturated fatty acid, is a reversible post-translational modification responsible for the regulation of numerous biological mechanisms. We found that LLC-PK<sub>1</sub> NET is dynamically palmitoylated, and that inhibition with the palmitoyl acyltransferase (DHHC) inhibitor, 2-bromopalmitate (2BP) results in decreased NET palmitoylation within 90 min of treatment. This result was followed closely by a reduction in transport capacity, cell surface, and total cellular NET expression after 120 min of treatment. Increasing 2BP concentrations and treatment time revealed a nearly complete loss of total NET protein. Co-expression with individual DHHCs revealed a single DHHC enzyme, DHHC1, promoted wild-type (WT) hNET palmitoylation and elevated NET protein levels. The POTS-associated NET mutant, A457P, exhibits dramatically decreased transport capacity and cell surface levels which we have confirmed in the current study. In an attempt to recover A457P NET expression, we co-expressed the A457P variant with DHHC1 to drive expression as seen with the WT protein but instead saw an increase in NET N-terminal immuno-detectable forms and fragments. Elimination of a potential palmitoylation site at cysteine 44 in the N-terminal tail of hNET resulted in a low expression phenotype mimicking the A457P hNET variant. Further investigation of A457P NET palmitoylation and surface expression is necessary, but our preliminary novel findings reveal palmitoylation as a mechanism of NET regulation and suggest that dysregulation of this process may contribute to the pathogenesis of adrenergic disorders like POTS.</p>\",\"PeriodicalId\":16527,\"journal\":{\"name\":\"Journal of Neurochemistry\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":4.2000,\"publicationDate\":\"2024-10-12\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of Neurochemistry\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1111/jnc.16241\",\"RegionNum\":3,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Neurochemistry","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1111/jnc.16241","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
摘要
体位性正位性心动过速综合征(POTS)是一种肾上腺素能信号紊乱,其特征是血浆去甲肾上腺素过多、体位性心动过速和晕厥。去甲肾上腺素转运体(NET)通过对细胞外儿茶酚胺的再摄取调节肾上腺素能平衡,并与肾上腺素能和神经系统疾病的发病机制有关。在这项研究中,我们发现在雄性 Sprague-Dawley 大鼠和 Lilly 实验室细胞猪肾(LLC-PK1)细胞中,NET 存在棕榈酰化。S-棕榈酰化或添加 16 碳饱和脂肪酸是一种可逆的翻译后修饰,负责调节多种生物机制。我们发现,LLC-PK1 NET 是动态棕榈酰化的,使用棕榈酰酰基酰基转移酶(DHHC)抑制剂 2-溴棕榈酸酯(2BP)进行抑制,可在 90 分钟内减少 NET 的棕榈酰化。紧接着,在处理 120 分钟后,转运能力、细胞表面和细胞 NET 总表达量也随之降低。随着 2BP 浓度和处理时间的增加,NET 蛋白总量几乎完全丧失。与单个 DHHC 的共表达显示,单个 DHHC 酶(DHHC1)促进了野生型(WT)hNET 棕榈酰化和 NET 蛋白水平的升高。POTS相关的NET突变体A457P的转运能力和细胞表面水平显著下降,我们在本研究中证实了这一点。为了恢复 A457P NET 的表达,我们将 A457P 变体与 DHHC1 共同表达,以驱动 WT 蛋白的表达,但结果却发现 NET N 端可免疫检测的形式和片段增加了。消除 hNET N 端尾部半胱氨酸 44 处的潜在棕榈酰化位点会导致模仿 A457P hNET 变体的低表达表型。有必要对 A457P NET 的棕榈酰化和表面表达进行进一步研究,但我们的初步新发现揭示了棕榈酰化是 NET 的一种调控机制,并表明这一过程的失调可能会导致肾上腺素能紊乱(如 POTS)的发病机制。
Palmitoylation regulates norepinephrine transporter uptake, surface localization, and total expression with pathogenic implications in postural orthostatic tachycardia syndrome.
Postural orthostatic tachycardia syndrome (POTS) is an adrenergic signaling disorder characterized by excessive plasma norepinephrine, postural tachycardia, and syncope. The norepinephrine transporter (NET) modulates adrenergic homeostasis via the reuptake of extracellular catecholamines and is implicated in the pathogenesis of adrenergic and neurological disorders. In this study, we reveal NET is palmitoylated in male Sprague-Dawley rats and Lilly Laboratory Cell Porcine Kidney (LLC-PK1) cells. S-palmitoylation, or the addition of a 16-carbon saturated fatty acid, is a reversible post-translational modification responsible for the regulation of numerous biological mechanisms. We found that LLC-PK1 NET is dynamically palmitoylated, and that inhibition with the palmitoyl acyltransferase (DHHC) inhibitor, 2-bromopalmitate (2BP) results in decreased NET palmitoylation within 90 min of treatment. This result was followed closely by a reduction in transport capacity, cell surface, and total cellular NET expression after 120 min of treatment. Increasing 2BP concentrations and treatment time revealed a nearly complete loss of total NET protein. Co-expression with individual DHHCs revealed a single DHHC enzyme, DHHC1, promoted wild-type (WT) hNET palmitoylation and elevated NET protein levels. The POTS-associated NET mutant, A457P, exhibits dramatically decreased transport capacity and cell surface levels which we have confirmed in the current study. In an attempt to recover A457P NET expression, we co-expressed the A457P variant with DHHC1 to drive expression as seen with the WT protein but instead saw an increase in NET N-terminal immuno-detectable forms and fragments. Elimination of a potential palmitoylation site at cysteine 44 in the N-terminal tail of hNET resulted in a low expression phenotype mimicking the A457P hNET variant. Further investigation of A457P NET palmitoylation and surface expression is necessary, but our preliminary novel findings reveal palmitoylation as a mechanism of NET regulation and suggest that dysregulation of this process may contribute to the pathogenesis of adrenergic disorders like POTS.
期刊介绍:
Journal of Neurochemistry focuses on molecular, cellular and biochemical aspects of the nervous system, the pathogenesis of neurological disorders and the development of disease specific biomarkers. It is devoted to the prompt publication of original findings of the highest scientific priority and value that provide novel mechanistic insights, represent a clear advance over previous studies and have the potential to generate exciting future research.