Drafting the CLN3 Protein Interactome in SH-SY5Y Human Neuroblastoma Cells: A Label-free Quantitative Proteomics Approach

Neuronal
ceroid lipofuscinoses (NCL) are the most common inherited
progressive encephalopathies of childhood. One of the most prevalent
forms of NCL, Juvenile neuronal ceroid lipofuscinosis (JNCL) or CLN3
disease (OMIM: 204200), is caused by mutations in the CLN3 gene on chromosome 16p12.1. Despite progress in the NCL field, the
primary function of ceroid-lipofuscinosis neuronal protein 3 (CLN3)
remains elusive. In this study, we aimed to clarify the role of human
CLN3 in the brain by identifying CLN3-associated proteins using a
Tandem Affinity Purification coupled to Mass Spectrometry (TAP-MS)
strategy combined with Significance Analysis of Interactome (SAINT).
Human SH-SY5Y-NTAP-CLN3 stable cells were used to isolate native protein
complexes for subsequent TAP-MS. Bioinformatic analyses of isolated
complexes yielded 58 CLN3 interacting partners (IP) including 42 novel
CLN3 IP, as well as 16 CLN3 high confidence interacting partners (HCIP)
previously identified in another high-throughput study by Behrends
et al., 2010. Moreover, 31 IP of ceroid-lipofuscinosis neuronal protein
5 (CLN5) were identified (18 of which were in common with the CLN3
bait). Our findings support previously suggested involvement of CLN3
in transmembrane transport, lipid homeostasis and neuronal excitability,
as well as link it to G-protein signaling and protein folding/sorting
in the ER.

神经元蜡样脂褐质沉积症（Neuronal ceroid lipofuscinoses, NCL）是儿童最常见的遗传性进行性脑病。其中最常见的亚型之一为青少年型神经元蜡样脂褐质沉积症（Juvenile neuronal ceroid lipofuscinosis, JNCL），或称CLN3病（OMIM: 204200），其致病原因为16号染色体16p12.1区域的CLN3基因发生突变。尽管NCL领域的研究已取得诸多进展，但蜡样脂褐质沉积症神经元蛋白3（ceroid-lipofuscinosis neuronal protein 3, CLN3）的核心生理功能仍未明确。本研究旨在阐明人类CLN3在脑组织中的作用机制，通过采用串联亲和纯化联合质谱（Tandem Affinity Purification coupled to Mass Spectrometry, TAP-MS）策略，并结合相互作用组显著性分析（Significance Analysis of Interactome, SAINT）筛选CLN3的相互作用蛋白。研究使用人类SH-SY5Y-NTAP-CLN3稳定细胞株分离天然蛋白质复合物，以开展后续的TAP-MS实验。对分离得到的复合物进行生物信息学分析后，共鉴定出58个CLN3相互作用蛋白（interacting partner, IP），其中包括42个全新的CLN3相互作用蛋白，以及16个由Behrends等人于2010年在另一项高通量研究中鉴定得到的高置信度CLN3相互作用蛋白（high confidence interacting partner, HCIP）。此外，本研究还鉴定出31个蜡样脂褐质沉积症神经元蛋白5（ceroid-lipofuscinosis neuronal protein 5, CLN5）的相互作用蛋白，其中18个与CLN3诱饵蛋白的筛选结果重合。本研究结果不仅支持了此前提出的CLN3参与跨膜运输、脂质稳态及神经元兴奋性调控的观点，还将其与G蛋白信号通路及内质网（endoplasmic reticulum, ER）中的蛋白质折叠与分选过程建立了关联。