Segal-Salto Michal, Hansson Karin, Sapir Tamar, Kaplan Anna, Levy Talia, Schweizer Michaela, Frotscher Michael, James Peter, Reiner Orly
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.
Department of Immunotechnology, Lund University, Medicon Village, Lund, Sweden and BTK, Åbo Academy University, Turku, Finland.
Hum Mol Genet. 2017 May 1;26(9):1678. doi: 10.1093/hmg/ddx074.
Mutations in the depalmitoylation enzyme, palmitoyl protein thioesterase (PPT1), result in the early onset neurodegenerative disease known as Infantile Neuronal Ceroid Lipofuscinosis. Here, we provide proteomic evidence suggesting that PPT1 deficiency could be considered as a ciliopathy. Analysis of membrane proteins from brain enriched for acylated proteins from neonate Ppt1 knock out and control mice revealed a list of 88 proteins with differential expression levels. Amongst them, we identified Rab3IP, which regulates ciliogenesis in concert with Rab8 and Rab11. Immunostaining analysis revealed that PPT1 is localized in the cilia. Indeed, an unbiased proteomics analysis on isolated cilia revealed 660 proteins, which differed in their abundance levels between wild type and Ppt1 knock out. We demonstrate here that Rab3IP, Rab8 and Rab11 are palmitoylated, and that palmitoylation of Rab11 is required for correct intracellular localization. Cells and brain preparations from Ppt1-/- mice exhibited fewer cells with cilia and abnormally longer cilia, with both acetylated tubulin and Rab3IP wrongly distributed along the length of cilia. Most importantly, the analysis revealed a difference in the distribution and levels of the modified proteins in cilia in the retina of mutant mice versus the wildtype, which may be important in the early neurodegenerative phenotype. Overall, our results suggest a novel link between palmitoylated proteins, cilial organization and the pathophysiology of Neuronal Ceroid Lipofuscinosis.
去棕榈酰化酶棕榈酰蛋白硫酯酶（PPT1）的突变会导致早发性神经退行性疾病，即婴儿神经元蜡样脂褐质沉积症。在此，我们提供蛋白质组学证据表明PPT1缺乏可被视为一种纤毛病。对来自新生Ppt1基因敲除小鼠和对照小鼠的富含酰化蛋白的脑细胞膜蛋白进行分析，发现了88种表达水平存在差异的蛋白质。其中，我们鉴定出了Rab3IP，它与Rab8和Rab11协同调节纤毛发生。免疫染色分析表明PPT1定位于纤毛中。实际上，对分离出的纤毛进行的无偏向蛋白质组学分析揭示了660种蛋白质，其在野生型和Ppt1基因敲除小鼠之间的丰度水平有所不同。我们在此证明Rab3IP、Rab8和Rab11是棕榈酰化的，并且Rab11的棕榈酰化对于其正确的细胞内定位是必需的。来自Ppt1 - / - 小鼠的细胞和脑组织样本显示，有纤毛的细胞数量减少，纤毛异常变长，乙酰化微管蛋白和Rab3IP在纤毛长度上分布错误。最重要的是，分析揭示了突变小鼠与野生型小鼠视网膜纤毛中修饰蛋白的分布和水平存在差异，这可能对早期神经退行性表型很重要。总体而言，我们的结果表明棕榈酰化蛋白、纤毛组织与神经元蜡样脂褐质沉积症的病理生理学之间存在新的联系。
