PSEEN/PEN2是γ-分泌酶复合物的最小亚基,一种膜内蛋白酶,在蛋白质的跨膜结构域内切割蛋白质。γ-分泌酶组分的突变是家族性阿尔茨海默病的基础。除了它的蛋白水解活性,补充,γ-分泌酶非依赖性,已经提出了巨自噬/自噬-溶酶体系统中的功能。这里,我们筛选了PSEEN相互作用蛋白并鉴定了CLN3。CLN3的突变是幼年神经元类脂褐菌病的病因,一种罕见的溶酶体贮积症被认为是儿童最常见的神经退行性疾病。由于PSEEN和CLN3基因的突变会导致不同的神经退行性疾病,理解两种蛋白质共同的细胞功能可能与理解神经变性的一般细胞机制有关.我们假设CLN3调节γ-分泌酶活性,PSEEN和CLN3在自噬-溶酶体系统中发挥相关作用。我们应用CRISPR基因编辑并获得了PSEEN和CLN3的独立等基因HeLa敲除细胞系。根据以前的研究,我们证明了PSEEN对于形成功能性γ-分泌酶复合物是必需的,并且对于γ-分泌酶活性是必不可少的。相比之下,CLN3不显著调节γ-分泌酶活性。我们在PSENEN和CLN3敲除细胞中观察到自噬-溶酶体系统的相应改变。这些包括溶酶体酶和溶酶体数量的活性降低,自噬体数量增加,溶酶体-自噬体融合增加,和升高的TFEB(转录因子EB)水平。我们的研究强烈表明,PSEEN和CLN3在自噬-溶酶体系统中以不依赖γ-分泌酶活性的方式发挥着重要作用。支持不同神经退行性疾病的共同细胞病理学过程的想法。缩写:Aβ,淀粉样β;AD,阿尔茨海默病;APP,淀粉样前体蛋白;ATP5MC,ATP合酶膜亚基c;DQ-BSA,染料猝灭的牛血清白蛋白;ER,内质网;GFP,绿色荧光蛋白;ICC,免疫细胞化学;ICD,胞内结构域;JNCL,幼年神经元类脂褐菌病;KO,敲除;LC3,微管相关蛋白1轻链3;NCL,神经元类脂褐斑病;PSEN,早老素;PSEEN/PEN2:早老素增强剂,γ-分泌酶亚基;TAP,串联亲和纯化;TEV,烟草蚀刻病毒;TF,转铁蛋白;WB,Westernblot;WT,野生型。
PSENEN/PEN2 is the smallest subunit of the γ-secretase complex, an intramembrane protease that cleaves proteins within their transmembrane domains. Mutations in components of the γ-secretase underlie familial Alzheimer disease. In addition to its proteolytic activity, supplementary, γ-secretase independent, functions in the macroautophagy/autophagy-lysosome system have been proposed. Here, we screened for PSENEN-interacting proteins and identified CLN3. Mutations in CLN3 are causative for juvenile neuronal ceroid lipofuscinosis, a rare lysosomal storage disorder considered the most common neurodegenerative disease in children. As mutations in the PSENEN and CLN3 genes cause different neurodegenerative diseases, understanding shared cellular functions of both proteins might be pertinent for understanding general cellular mechanisms underlying neurodegeneration. We hypothesized that CLN3 modulates γ-secretase activity and that PSENEN and CLN3 play associated roles in the autophagy-lysosome system. We applied CRISPR gene-editing and obtained independent isogenic HeLa knockout cell lines for PSENEN and CLN3. Following previous studies, we demonstrate that PSENEN is essential for forming a functional γ-secretase complex and is indispensable for γ-secretase activity. In contrast, CLN3 does not modulate γ-secretase activity to a significant degree. We observed in PSENEN- and CLN3-knockout cells corresponding alterations in the autophagy-lysosome system. These include reduced activity of lysosomal enzymes and lysosome number, an increased number of autophagosomes, increased lysosome-autophagosome fusion, and elevated levels of TFEB (transcription factor EB). Our study strongly suggests converging roles of PSENEN and CLN3 in the autophagy-lysosome system in a γ-secretase activity-independent manner, supporting the idea of common cytopathological processes underlying different neurodegenerative diseases.Abbreviations: Aβ, amyloid-beta; AD, Alzheimer disease; APP, amyloid precursor protein; ATP5MC, ATP synthase membrane subunit c; DQ-BSA, dye-quenched bovine serum albumin; ER, endoplasmic reticulum; GFP, green fluorescent protein; ICC, immunocytochemistry; ICD, intracellular domain; JNCL, juvenile neuronal ceroid lipofuscinosis; KO, knockout; LC3, microtubule associated protein 1 light chain 3; NCL, neuronal ceroid lipofuscinoses; PSEN, presenilin; PSENEN/PEN2: presenilin enhancer, gamma-secretase subunit; TAP, tandem affinity purification; TEV, tobacco etch virus; TF, transferrin; WB, Western blot; WT, wild type.