Fig 1: Proposed model based on the findings.a Glucocorticoid-mediated stress induces lysosomal damage, which leads to the association of FKBP51 with the cargo receptor TRIM16 and the cargo protein (e.g., MMP9). This complex is transported on the autophagosome membrane via the interaction of FKBP51 with SEC22B. The cargo protein is internalized into the autophagosome which is transported to the plasma membrane. The vesicle-plasma membrane fusion is mediated via the SNARE-protein complex assembly, which is regulated by FKBP51 and sensitive to glucocorticoids. The membrane fusion leads to the release of the cargo proteins into the extracellular milieu. The increased secretion of extracellular MMP9 induces the cleavage of proBDNF to its mature form, which becomes the prevailing form in the extracellular space; b A first response to stress triggers lytic autophagy10. In case the stress persists, a second defense line is activated which switches the stress response from lytic to secretory autophagy. If stress further persists (e.g. chronic stress), the initially beneficial proteins secreted in response to stress, might lead to neuroinflammation and psychiatric disorders.
Fig 2: FKBP51 links stress to secretory autophagy.a Results of automated literature mining of FKBP51 interactors in association to “autophagy”, “proteostasis” and “ubiquitin-proteasome system” (UPS) or none of them. b Western blotting of FKBP51 and SEC22B in GFP-tagged SEC22B co-IP (GFP-IP) and whole-cell extract (WCE) as control; c Western blotting of FKBP51 and SEC22B in FLAG-tagged FKBP51 co-IP (FLAG-IP) and WCE as control. d Western blotting of FKBP51, TRIM16, LC3B, and CTSD in FLAG-tagged FKBP51 co-IP (FLAG-IP) and WCE as control. e Western blotting for TRIM16, CTSD, and SEC22B in FLAG-tagged TRIM16 co-IP (FLAG-IP) and WCE as control performed in WT and FKBP5 KO SH-SY5Y cells. f Quantifications of e with n = 3 biologically independent samples. g Western blotting of TRIM16, CTSD, and SEC22B in FLAG-tagged TRIM16 co-IP (FLAG-IP) and WCE as control performed in cells treated with 100 nM dexamethasone or vehicle for 4 h. h Quantifications of g with n = 3 biologically independent samples. i Western blotting for FKBP51, SNAP23, SNAP29, STX3, and STX4 in FLAG-tagged FKBP51 co-IP (FLAG-IP) and WCE as control. j Western blotting of SEC22B, SNAP23, SNAP29, STX3, and STX4 in GFP-tagged SEC22B co-IP (GFP-IP) and WCE as control performed in WT and FKBP5 KO cells treated with 100 nM dexamethasone or vehicle for 4 h. k Quantifications of j with n = 3 biologically independent samples. b–k All experiments were performed in SH-SY5Y cells. l Schematic model of the interactions of FKBP51 in the secretory autophagy pathway. Unpaired, one-tailed t-tests were performed for all quantifications; ns not significant, **P < 0.01, ***P < 0.001. Data shown as mean ± s.e.m. Ab antibody, Fc fold change.
Fig 3: E-Syt1 is required in PKCd secretion in liver cancer cell lines. (A) Immunoblot analysis of lysates or media from starved control or E-Syt1 KO HepG2 cells for 24 h; n = 3 independent experiments. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and AFP were used as a loading control. (B) Immunoblot analysis of lysates and media (24 h) from starved HuH7 cells treated with scrambled (Scr) or E-Syt1 siRNA; n = 3 independent experiments. AFP was used as a loading control. (C) PKCd secretion measured by HiBiT extracellular assay in doxycycline-inducible HepG2 cells treated with scrambled (Scr), E-Syt1, SEC22B, or STX3 siRNA for 24 h in 10% FBS condition; n = 4 independent experiments. Luminescence was measured after cells were re-cultured in a medium containing 0.5 µg/mL doxycycline for 24 h. Data are shown as the mean ± s.d. One-way ANOVA, p = 0.002 with post-test Bonferroni’s test with pairwise comparison with siScr (siE-Syt1, * p < 0.0001; siSEC22B, ** p = 0.0007; and siSTX3, *** p = 0.0041). (D) Confocal micrographs to detect the interaction between SEC22B and STX3 (a SNARE on the PM) in control or E-Syt1 KO HepG2 cells. Each cell was fixed, reacted with a combination of mouse anti-SEC22B and rabbit anti-STX3 antibodies (SEC22B × STX3), and subjected to Duolink in situ PLA. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Data are shown as the mean ± s.d., * p < 0.0001 (two-tailed Student’s t-test) (n = 49 for control HepG2 cells and n = 52 for E-Syt1 KO HepG2 cells). Images represent three independent experiments. Scale bars, 10 µm.
Fig 4: Other cytoplasmic proteins are secreted via the same autophagy-related system. (A) Confocal micrographs showing colocalization of endogenous importin a1 with PDI or Calnexin in HepG2 cells. Images are representative of three independent experiments. (Scale bars, 10 µm.) (Insets) Magnified views of the regions in the white boxes, Upper: (x 10), Lower: (x 5). (B) Confocal micrographs showing colocalization of endogenous NCL with ER using ERseeing in HepG2 cells. Images are representative of three independent experiments. White arrows indicate colocalization of NCL with the ER. (Scale bar, 10 µm.) (C) Importin a1 secretion measured by HiBiT extracellular assay in doxycycline-inducible HepG2 cells treated with scrambled (Scr), E-Syt1, ATG5, LC3, or SEC22B siRNAs (2 nM) for 48 h; n = 4 independent experiments. Luminescence was measured after cells recultured with medium containing 1 µg/mL doxycycline for 24 h. Data are shown as means ± SD, *P < 0.0002; **P = 0.0018; ***P = 0.0104; ****P = 0.0001 (ANOVA). (D) NCL secretion measured by HiBiT extracellular assay in doxycycline-inducible HepG2 cells treated with scrambled (Scr), E-Syt1, ATG5, LC3, or SEC22B siRNAs (2 nM) for 48 h; n = 4 independent experiments. Luminescence was measured after cells recultured with medium containing 1 µg/mL doxycycline for 24 h. Data are shown as means ± SD, *P < 0.001; **P = 0.0022; ***P = 0.0007; ****P = 0.0003 (ANOVA). (E) Confocal micrographs to detect the interaction with importin a1 and indicated proteins in HepG2 cells. Cells are fixed, reacted with combination of mouse anti-importin a1 or LC3B and rabbit antibodies, and subjected to Duolink in situ PLA. The images are representative of two independent experiments. (Scale bars, 10 µm.) (F) Confocal micrographs to detect the interaction with NCL and LC3B or SEC22B in HepG2 cells. Cells are fixed, reacted with combination of mouse anti-LC3B or SEC22B and a rabbit anti-NCL antibody, and subjected to Duolink in situ PLA. The images are representative of two independent experiments. (Scale bars, 10 µm.) (G) A model for the role of E-Syt1 as a link between the ER and vesicle-mediated secretion of cytosolic proteins. Packaging of cytosolic proteins into SEC22B+ vesicles is apparently dependent on the expression of E-Syt1 and autophagy-related proteins. E-Syt1 may form a specific complex for cytosolic protein secretion at the ER in liver cancer cells.
Fig 5: PKCd is secreted via SEC22B+ vesicle. (A) PKCd secretion measured by HiBiT extracellular assay in doxycycline-inducible HepG2 cells treated with scrambled (Scr) or SEC22B siRNAs (2 nM) for 48 h; n = 3; independent experiments. Luminescence was measured after cells were recultured with a medium containing 0.5 µg/mL doxycycline for 24 h. Data are shown as mean ± SD, *P = 0.0223 (two-tailed Student’s t test). (B) PKCd secretion measured by HiBiT extracellular assay in doxycycline-inducible SEC22B KO HepG2 cells; n = 3; independent experiments. Luminescence was measured after cells were recultured with a medium containing 0.5 µg/mL doxycycline for 24 h. Data are shown as mean ± SD, *P = 0.0002 (two-tailed Student’s t test). (C) Confocal micrographs to indicate the proximity with PKCd and SEC22B in parental HepG2 (control), E-Syt1 KO HepG2 (E-Syt1 KO), or AGS cells. Each cell is fixed, reacted with a combination of mouse anti-PKCd and rabbit anti-SEC22B antibodies (PKCd × SEC22B), and subjected to Duolink in situ PLA. Data are shown as mean ± SD (n = 20 for control HepG2 cells and n = 31 for E-Syt1 KO HepG2 cells, and n = 20 for AGS WT cells), *P < 0.0001 (two-tailed Mann–Whitney test). Images are representative of three independent experiments. (Scale bars, 10 µm.) (D) PKCd secretion measured by HiBiT extracellular assay in doxycycline-inducible HepG2 cells treated with scrambled (Scr) or GRASP55 siRNAs (2 nM) for 48 h; n = 4 independent experiments. Luminescence was measured after cells were recultured with normal 10% FBS-containing medium or EBSS with 0.5 µg/mL doxycycline for 24 h. Data are shown as mean ± SD, *P = 0.0027, **P = 0.0112, ***P = 0.0061 (ANOVA), n.s., not significant. (E) Electron micrographs of HepG2 cells showing the existence of PKCd (arrows) in SEC22B+ (arrowheads) vesicles at the vicinity of the PM (Left) and moment of the vesicle-PM fusion and secretion of PKCd (Right). Images are representative and two similar independent experiments were performed. (Scale bars, 100 nm.) (F) PKCd secretion measured by HiBiT extracellular assay in doxycycline-inducible HepG2 cells treated with scrambled (Scr), SNAP23, STX3, STX4, or a mixture with STX3 and STX4 siRNAs (2 nM) for 48 h; n > 3; independent experiments. Luminescence was measured after the cells were recultured with a medium containing 0.5 µg/mL doxycycline for 24 h. Data are shown as mean ± SD, *P < 0.0001 (ANOVA). (G) Images of tumor tissues of patients with hepatocellular carcinoma to indicate tumor cell-specific colocalization of PKCd with SEC22B using Duolink in situ PLA (Center and Right). Tumor (T) and nontumor (NT) lesion were defined by evaluating H&E staining of each tumor section (Left). Two lines of images are representative in sections of five patients with liver cancer. (Scale bars, 50 µm.) (H) Confocal micrographs to detect the interaction with PKCd and SEC22B or Stx17 in HepG2 cells treated with DMSO or BafA1 (100 nM) for 6 h. Each Cell is fixed, reacted with a combination of mouse anti-PKCd and rabbit anti-SEC22B or Stx17 antibodies (PKCd x SEC22B or PKCd × Stx17), and subjected to Duolink in situ PLA. Data are shown as mean ± SD (PKCd × SEC22B; n = 30 for DMSO-treated and n = 30 for BafA1-treated cells, PKCd × Stx17; n = 43 for DMSO-treated and n = 44 for BafA1-treated cells,), *P < 0.0001 (two-tailed Student’s test), n.s., not significant. Images are representative of two independent experiments. (Scale bars, 10 µm.)
Supplier Page from Abcam for Anti-SEC22B antibody [EPR12335]