Author: Lin, Mingqun; Liu, Hongyan; Xiong, Qingming; Niu, Hua; Cheng, Zhihui; Yamamoto, Akitsugu; Rikihisa, Yasuko
Title: Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase Document date: 2016_8_19
ID: x5y551c8_30
Snippet: RAB GTPase cycles between GTP-and GDP-bound forms and acts via RAB effectors. 102,103 RAB effectors such as PIK3C3 and ANKFY1 bind to RAB-GTP but dissociate from RAB-GDP. 45 ,95 RAB5A S34N (dominant-negative RAB5A/ RAB5A-DN), a GDP-bound form of RAB5 that sequesters RAB5 guanine nucleotide exchange factor (RAB5-GEF) and thus prevents RAB5 activation. 104 In RAB5-regulated autophagy, RAB5-DN decreases LC3-puncta formation and increases ATG5-contai.....
Document: RAB GTPase cycles between GTP-and GDP-bound forms and acts via RAB effectors. 102,103 RAB effectors such as PIK3C3 and ANKFY1 bind to RAB-GTP but dissociate from RAB-GDP. 45 ,95 RAB5A S34N (dominant-negative RAB5A/ RAB5A-DN), a GDP-bound form of RAB5 that sequesters RAB5 guanine nucleotide exchange factor (RAB5-GEF) and thus prevents RAB5 activation. 104 In RAB5-regulated autophagy, RAB5-DN decreases LC3-puncta formation and increases ATG5-containing structures (autophagosome precursors). 97 To analyze whether RAB5 GTPase cycle is involved in Etf-1 interaction with RAB5 and the BECN1-class III PtdIns3K complex, and Etf-1-induced autophagy, Etf-1-HA and GFP vector control, GFP-RAB5A (WT), GFP-RAB5A-DN, or GFP-RAB5A Q79L (constitutively active RAB5A/RAB5A-CA) were cotransfected into HEK293 cells, and the cell lysates were immunoprecipitated with anti-HA. Western blot analysis showed that although Etf-1-GFP bound RAB5A (WT), RAB5A-DN and RAB5A-CA, PIK3C3 and BECN1 interacted with Etf-1 only in the presence of RAB5A (WT) and RAB5A-CA (Fig. 11A) . Fluorescence microscopy showed enlarged GFP-RAB5A-CA endosomes that colocalized with Etf-1 puncta (Fig. 11B) . Colocalization analysis between GFP-RAB5A-CA with Etf-1 showed that the Pearson correlation coefficient of 24 § 2 %, which was increased to 49 § 5% when cells were treated with rapamycin for 16 h before harvesting at 2 d post-transfection (p.t.), and both were significantly greater (P < 0.01) vs. GFP control (12 § 2 %) by the Student unpaired t test. Taken together, Etf-1 interacts with the RAB5-BECN1-PIK3C3, autophagy master regulator complex via GTP-bound RAB5, to induce autophagy. Overexpression of GFP-RAB5A-DN impaired E. chaffeensis infection compared with cells transfected with GFP-RAB5A (WT) or GFP-RAB5A-CA (Fig. 11C) , indicating that activated RAB5 (the GTP-bound state of RAB5) is indeed critical for infection. RABs have a low intrinsic rate of GTP hydrolysis, which is controlled by GAPs/GTPase activating proteins. Thus we assessed the effects of a RAB5-specific GAP, SGSM3/RAB-GAP5 (small G protein signaling modulator 3), 105 on E. chaffeensis infection. SGSM3 overexpression at 1 d p.i. profoundly impaired E. chaffeensis replication compared with overexpression of SGSM3 R165A (SGSM3-RA), a catalytic site mutant 105 (Fig. 11C) , confirming the critical role of GTP-bound RAB5 in E. chaffeensis replication. Taken together, Etf-1 induces autophagy by recruiting the BECN1-class III PtdIns3K complex via GTP-bound RAB5, to promote E. chaffeensis replication.
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