Figure 9.
NRBF2 regulates APP-CTF degradation via CCZ1-MON1A-mediated RAB7 activation. (A-D) Overexpression of WT, RAB7Q67L and RAB7T22N in nrbf2-/- N2S cells and measurement of FL-APP and APP-CTFs by WB. Data are quantified as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. the relative control. (E and F) ELISA results demonstrated that active RAB7 overexpression significantly reduced intracellular Aβ. Data are quantified as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. the relative control. (G) Endogenous APP interacts with CCZ1-MON1A. N2S cells were lysed and immunoprecipitated (IP) with APP antibody; eluates were resolved by SDS-PAGE and analyzed by immunoblotting with the CCZ1 antibodies. (H) CCZ1 interacts with APP in N2S cells. N2S cells were lysed and immunoprecipitated (IP) with CCZ1 antibody; eluates were resolved by SDS-PAGE and analyzed by immunoblotting with the APP antibodies. (I) Flag-MON1A interacts with APP in N2S cells. FLAG-MON1A were transfected into N2S, then N2S cells were lysed and immunoprecipitated (IP) with Flag antibody; eluates were resolved by SDS-PAGE and analyzed by immunoblotting with the APP antibodies. (J) FLAG-CCZ1 and FLAG-MON1A were transfected into N2a cells together with GFP-APP, the colocalization of Flag-CCZ1 or GFP-APP and Flag-MON1A or GFP-APP were visualized under confocal microscope. Scale bar: 5 μm. (K) APP-associated GEF activity was significantly reduced in nrbf2-/- N2S cells. (L-N) Interaction between APP and CCZ1 or RAB7 was determined by IP in WT and nrbf2-/- N2S cells. nrbf2 KO impaired interaction between CCZ1 and APP, RAB7. (O) Schematic model for the NRBF2 function in regulating autophagosome maturation. NRBF2 may serve as an enhancer for PtdIns3K complex I and MON1A-CCZ1 complex association to activate the GEF activity and subsequent RAB7 activation on the autophagosome, so as to promote the autophagosome maturation