Scifo Enzo, Szwajda Agnieszka, Soliymani Rabah, Pezzini Francesco, Bianchi Marzia, Dapkunas Arvydas, Dębski Janusz, Uusi-Rauva Kristiina, Dadlez Michał, Gingras Anne-Claude, Tyynelä Jaana, Simonati Alessandro, Jalanko Anu, Baumann Marc H, Lalowski Maciej
Meilahti Clinical Proteomics Core Facility, Institute of Biomedicine/Biochemistry and Developmental Biology, University of Helsinki, Helsinki, Finland; Doctoral Program Brain & Mind, University of Helsinki, Helsinki, Finland.
Institute for Molecular Medicine (FIMM), University of Helsinki, Helsinki, Finland.
J Proteomics. 2015 Jun 18;123:42-53. doi: 10.1016/j.jprot.2015.03.038. Epub 2015 Apr 10.
Neuronal ceroid lipofuscinoses (NCL) are a group of inherited progressive childhood disorders, characterized by early accumulation of autofluorescent storage material in lysosomes of neurons or other cells. Clinical symptoms of NCL include: progressive loss of vision, mental and motor deterioration, epileptic seizures and premature death. CLN1 disease (MIM#256730) is caused by mutations in the CLN1 gene, which encodes palmitoyl protein thioesterase 1 (PPT1). In this study, we utilised single step affinity purification coupled to mass spectrometry (AP-MS) to unravel the in vivo substrates of human PPT1 in the brain neuronal cells. Protein complexes were isolated from human PPT1 expressing SH-SY5Y stable cells, subjected to filter-aided sample preparation (FASP) and analysed on a Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer. A total of 23 PPT1 interacting partners (IP) were identified from label free quantitation of the MS data by SAINT platform. Three of the identified PPT1 IP, namely CRMP1, DBH, and MAP1B are predicted to be palmitoylated. Our proteomic analysis confirmed previously suggested roles of PPT1 in axon guidance and lipid metabolism, yet implicates the enzyme in novel roles including: involvement in neuronal migration and dopamine receptor mediated signalling pathway.
The significance of this work lies in the unravelling of putative in vivo substrates of human CLN1 or PPT1 in brain neuronal cells. Moreover, the PPT1 IP implicate the enzyme in novel roles including: involvement in neuronal migration and dopamine receptor mediated signalling pathway.
神经元蜡样脂褐质沉积症（NCL）是一组遗传性进行性儿童疾病，其特征是自荧光储存物质在神经元或其他细胞的溶酶体中早期积累。NCL的临床症状包括：视力逐渐丧失、智力和运动功能退化、癫痫发作和过早死亡。CLN1疾病（MIM#256730）由CLN1基因突变引起，该基因编码棕榈酰蛋白硫酯酶1（PPT1）。在本研究中，我们利用单步亲和纯化结合质谱（AP-MS）来揭示人脑神经元细胞中人类PPT1的体内底物。从表达人类PPT1的SH-SY5Y稳定细胞中分离蛋白质复合物，进行过滤辅助样品制备（FASP），并在Q Exactive杂交四极杆-轨道阱质谱仪上进行分析。通过SAINT平台对MS数据进行无标记定量，共鉴定出23个PPT1相互作用伙伴（IP）。鉴定出的三个PPT1 IP，即CRMP1、DBH和MAP1B，预计会被棕榈酰化。我们的蛋白质组学分析证实了PPT1先前在轴突导向和脂质代谢中的作用，但也表明该酶参与了新的作用，包括：参与神经元迁移和多巴胺受体介导的信号通路。
这项工作的意义在于揭示了人脑神经元细胞中人类CLN1或PPT1的假定体内底物。此外，PPT1 IP表明该酶参与了新的作用，包括：参与神经元迁移和多巴胺受体介导的信号通路。
