Down-regulated TMED10 in Alzheimer disease induces autophagy via ATG4B activation

Several studies have shown that dysfunction of macroautophagy/autophagy is associated with many human diseases, including neurodegenerative disease and cancer. To explore the molecular mechanisms of autophagy, we performed a cell-based functional screening with SH-SY5Y cells stably expressing GFP-LC3, using an siRNA library and identified TMED10 (transmembrane p24 trafficking protein 10), previously known as the γ-secretase-modulating protein, as a novel regulator of autophagy. Further investigations revealed that depletion of TMED10 induced the activation of autophagy. Interestingly, protein-protein interaction assays showed that TMED10 directly binds to ATG4B (autophagy related gene 4B cysteine peptidase), and the interaction is diminished under autophagy activation conditions such as rapamycin treatment and serum deprivation. In addition, inhibition of TMED10 significantly enhanced the proteolytic activity of ATG4B for LC3 cleavage. Importantly, the expression of TMED10 in AD (Alzheimer disease) patients was considerably decreased, and downregulation of TMED10 increased amyloid-β (Aβ) production. Treatment with Aβ increased ATG4B proteolytic activity as well as dissociation of TMED10 and ATG4B. Taken together, our results suggest that the AD-associated protein TMED10 negatively regulates autophagy by inhibiting ATG4B activity.<b>Abbreviations</b>: Aβ: amyloid-β; AD: Alzheimer disease; ATG: autophagy related; BECN1: beclin 1; BiFC: bimolecular fluorescence complementation; CD: cytosolic domain; GFP: green fluorescent protein; GLUC: <i>Gaussia</i> luciferase; IP: immunoprecipitation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LD: luminal domain; PD: Parkinson disease; ROS: reactive oxygen species; siRNA: small interfering RNA; SNP: single-nucleotide polymorphisms; TD: transmembrane domain; TMED10: transmembrane p24 trafficking protein 10; VC: C terminus of Venus fluorescent protein; VN: N terminus of Venus fluorescent protein.

多项研究证实，巨自噬（macroautophagy，通常简称自噬）功能异常与诸多人类疾病密切相关，涵盖神经退行性疾病及癌症。为探究自噬的分子机制，我们以稳定表达GFP-LC3融合蛋白的SH-SY5Y细胞为模型，借助小干扰RNA（small interfering RNA，siRNA）文库开展基于细胞的功能筛选，最终鉴定出跨膜p24转运蛋白10（transmembrane p24 trafficking protein 10，TMED10）——此前被称为γ分泌酶调控蛋白——作为一种新型自噬调控因子。进一步研究发现，敲低TMED10可诱导自噬激活。有趣的是，蛋白质相互作用实验证实，TMED10可直接结合自噬相关基因4B半胱氨酸肽酶（autophagy related gene 4B cysteine peptidase，ATG4B），且在雷帕霉素处理、血清剥夺等自噬激活条件下，二者的相互作用会显著减弱。此外，抑制TMED10可显著增强ATG4B对LC3的蛋白水解活性。值得关注的是，阿尔茨海默病（Alzheimer disease，AD）患者体内TMED10的表达水平显著降低，而TMED10的下调会促进淀粉样β（amyloid-β，Aβ）的生成。Aβ处理可同时提升ATG4B的蛋白水解活性，并加速TMED10与ATG4B的解离。综上，本研究结果表明，与阿尔茨海默病相关的TMED10可通过抑制ATG4B的活性，负向调控自噬。**缩写说明**：Aβ：淀粉样β（amyloid-β）；AD：阿尔茨海默病（Alzheimer disease）；ATG：自噬相关（autophagy related）；BECN1：Beclin 1；BiFC：双分子荧光互补（bimolecular fluorescence complementation）；CD：胞质结构域（cytosolic domain）；GFP：绿色荧光蛋白（green fluorescent protein）；GLUC：高斯荧光素酶（Gaussia luciferase）；IP：免疫沉淀（immunoprecipitation）；MAP1LC3/LC3：微管相关蛋白1轻链3（microtubule associated protein 1 light chain 3）；LD：管腔结构域（luminal domain）；PD：帕金森病（Parkinson disease）；ROS：活性氧（reactive oxygen species）；siRNA：小干扰RNA（small interfering RNA）；SNP：单核苷酸多态性（single-nucleotide polymorphisms）；TD：跨膜结构域（transmembrane domain）；TMED10：跨膜p24转运蛋白10（transmembrane p24 trafficking protein 10）；VC：Venus荧光蛋白C端（C terminus of Venus fluorescent protein）；VN：Venus荧光蛋白N端（N terminus of Venus fluorescent protein）。