ABSTRACT

NRBF2 is a component of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex. Our previous study has revealed its role in regulating ATG14-associated PtdIns3K activity for autophagosome initiation. In this study, we revealed an unknown mechanism by which NRBF2 modulates autophagosome maturation and APP-C-terminal fragment (CTF) degradation. Our data showed that NRBF2 localized at autolysosomes, and loss of NRBF2 impaired autophagosome maturation. Mechanistically, NRBF2 colocalizes with RAB7 and is required for generation of GTP-bound RAB7 by interacting with RAB7 GEF CCZ1-MON1A and maintaining the GEF activity. Specifically, NRBF2 regulates CCZ1-MON1A interaction with PI3KC3/VPS34 and CCZ1-associated PI3KC3 kinase activity, which are required for CCZ1-MON1A GEF activity. Finally, we showed that NRBF2 is involved in APP-CTF degradation and amyloid beta peptide production by maintaining the interaction between APP and the CCZ1-MON1A-RAB7 module to facilitate the maturation of APP-containing vesicles. Overall, our study revealed a pivotal role of NRBF2 as a new RAB7 effector in modulating autophagosome maturation, providing insight into the molecular mechanism of NRBF2-PtdIns3K in regulating RAB7 activity for macroautophagy/autophagy maturation and Alzheimer disease-associated protein degradation..

Abbreviations: 3xTg AD, triple transgenic mouse for Alzheimer disease; Aβ, amyloid beta peptide; Aβ_1-40, amyloid beta peptide 1–40; Aβ_1-42, amyloid beta peptide 1–42; AD, Alzheimer disease; APP, amyloid beta precursor protein; APP-CTFs, APP C-terminal fragments; ATG, autophagy related; ATG5, autophagy related 5; ATG7, autophagy related 7; ATG14, autophagy related 14; CCD, coiled-coil domain; CCZ1, CCZ1 homolog, vacuolar protein trafficking and biogenesis associated; CHX, cycloheximide; CQ, chloroquine; DAPI, 4ʹ,6-diamidino-2-phenylindole; dCCD, delete CCD; dMIT, delete MIT; FYCO1, FYVE and coiled-coil domain autophagy adaptor 1; FYVE, Fab1, YGL023, Vps27, and EEA1; GAP, GTPase-activating protein; GDP, guanine diphosphate; GEF, guanine nucleotide exchange factor; GTP, guanine triphosphate; GTPase, guanosine triphosphatase; HOPS, homotypic fusion and vacuole protein sorting; ILVs, endosomal intralumenal vesicles; KD, knockdown; KO, knockout; LAMP1, lysosomal associated membrane protein 1; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MLVs, multilamellar vesicles; MON1A, MON1 homolog A, secretory trafficking associated; NRBF2, nuclear receptor binding factor 2; PtdIns3K, class III phosphatidylinositol 3-kinase; PtdIns3P, phosphatidylinositol-3-phosphate; RILP, Rab interacting lysosomal protein; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SQSTM1/p62, sequestosome 1; UVRAG, UV radiation resistance associated; VPS, vacuolar protein sorting; WT, wild type.

摘要

NRBF2 是 III 类磷脂酰肌醇 3-激酶 (PtdIns3K) 复合物的组成部分。我们之前的研究揭示了其在调节 ATG14 相关 PtdIns3K 活性以促进自噬体启动中的作用。在这项研究中，我们揭示了 NRBF2 调节自噬体成熟和 APP-C 末端片段 (CTF) 降解的未知机制。我们的数据显示 NRBF2 定位于自噬溶酶体，并且 NRBF2 的缺失会损害自噬体的成熟。从机制上讲，NRBF2 与 RAB7 共定位，并且是通过与 RAB7 GEF CCZ1-MON1A 相互作用并维持 GEF 活性来生成 GTP 结合的 RAB7 所必需的。具体而言，NRBF2 调节 CCZ1-MON1A 与 PI3KC3/VPS34 和 CCZ1 相关 PI3KC3 激酶活性的相互作用，这是 CCZ1-MON1A GEF 活性所必需的。最后，我们表明 NRBF2 通过维持 APP 和 CCZ1-MON1A-RAB7 模块之间的相互作用来促进含 APP 囊泡的成熟，从而参与 APP-CTF 降解和淀粉样蛋白 β 肽的产生。总的来说，我们的研究揭示了 NRBF2 作为一种新的 RAB7 效应物在调节自噬体成熟方面的关键作用，提供了对 NRBF2-PtdIns3K 在调节 RAB7 活性以促进巨自噬/自噬成熟和阿尔茨海默病相关蛋白质降解的分子机制的深入了解。

缩写： 3xTg AD，阿尔茨海默病三重转基因小鼠；Aβ，β-淀粉样肽；Aβ _1-40， β-淀粉样肽 1-40；Aβ _1-42，淀粉样蛋白 β 肽 1-42；AD，阿尔茨海默病；APP，β-淀粉样前体蛋白；APP-CTFs，APP C-末端片段；ATG，自噬相关；ATG5，自噬相关5；ATG7，自噬相关7；ATG14，自噬相关14；CCD，卷曲线圈域；CCZ1、CCZ1 同源物、液泡蛋白运输和生物发生相关；CHX，放线菌酮；CQ，氯喹；DAPI, 4ʹ,6-二脒基-2-苯基吲哚；dCCD，删除CCD；dMIT，删除MIT；FYCO1、FYVE 和卷曲螺旋结构域自噬适配器 1；FYVE、Fab1、YGL023、Vps27 和 EEA1；GAP，GTP酶激活蛋白；GDP，鸟嘌呤二磷酸；GEF，鸟嘌呤核苷酸交换因子；GTP，三磷酸鸟嘌呤；GTPase，鸟苷三磷酸酶；HOPS、同型融合和液泡蛋白分选；ILVs，内体腔内囊泡；KD，击倒；KO，淘汰赛；灯1，溶酶体相关膜蛋白 1；MAP1LC3/LC3，微管相关蛋白1轻链3；MLVs，多层囊泡；MON1A，MON1 同源物 A，分泌贩卖相关；NRBF2，核受体结合因子2；PtdIns3K，III 类磷脂酰肌醇 3-激酶；PtdIns3P，3-磷酸磷脂酰肌醇；RILP，Rab 相互作用溶酶体蛋白；圈套，可溶N-乙基马来酰亚胺敏感因子附着蛋白受体；SQSTM1/p62，螯合体 1；UVRAG，相关的抗紫外线辐射；VPS，液泡蛋白分选；WT，野生型。