11_20_07 Beckey

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  • - Other work - I have used SCAN MOTIF and ------ BECKY EXPLAIN
  • 11_20_07 Beckey

    1. 1. Ribosome-Mediated Stress Response Ribosome-Mediated Stress Response To Deoxynivalenol in the Macrophage Heekyong (Becky) Bae Food Science and Human Nutrition Center for Integrative Toxicology
    2. 2. Ribosome PKR Hck MAPK signaling pathway (p38, JNK, ERK) How does DON activate MAPK signaling pathway? DON ?
    3. 3. DON binding to the ribosome induces MAPK activation. <ul><ul><li>MAPK activation occur subsequently after DON uptake </li></ul></ul><ul><ul><li>When DON binding to the ribosome is inhibited by SG, MAPK activation by DON is inhibited. </li></ul></ul>
    4. 4. Design and Methods: Uptake of [ 3 H] DON RAW 264.7 cells Cell Lysate [ 3 H] DON 0, 250, 500, 1000 ng/ml 0, 2.5, 5, 10, 15, 30 min Scintillation counter % Maximum DON uptake Minutes Kinetics of [ 3 H] DON (250ng/ml) uptake into RAW 264.7 cells DON is taken up rapidly within 15 minutes. MAPK activation is also maximized within 15 minutes. 120 100 80 60 40 20 0 50 0 5 10 15 20 25 30 35 2.5
    5. 5. phopho-p38 phopho-JNK phopho-ERK 1 2 3 4 1: Vehicle 2: DON 5 min 3: DON 15 min 4: DON 30 min
    6. 6. SG(20ng/ml) was treated to Raw 264.7 cells for 1 hour and ribosomal fractions were separated using sucrose gradient system. Those fractions were reacted with additional SG and/or DON at 37 o C for 1 hour. Each groups was centrifuged and washed using Centricon tubes. 1: ribosomal fractions from SG treated cells 2: 1 + 20ng/ml SG 3: 1 + 500ng/ml DON 4: 1 + 20ng/ml SG + 500ng/ml DON Satratoxin binding sites of the ribosome were saturated and the binding was recovered by endogenous DON. a a a b a a a b
    7. 7. RAW 264.7 cells Phospho-p38 Phospho-JNK p38 JNK 1 2 3 4 1 2 3 4 1: Vehicle 2: SG 100ng/ml, 30min 3: DON 100ng/ml, 30min 4: SG+DON 100ng/ml, 30min
    8. 8. MAPK have direct interaction with the ribosome. <ul><ul><li>Motif scan predicts MAPK interaction with the ribosomal proteins. </li></ul></ul><ul><ul><li>MAPK interaction with the ribosome increases after DON treatment. </li></ul></ul>
    9. 9. http://Scansite.mit.edu Multiple ribosomal proteins might play scaffolding role for intracellular kinases Example: Ribosomal Protein L3
    10. 10. Ribosome fraction (Sucrose gradient separation) U937 cells DON Western Blotting 2 3 4 1 Monosome Polysome Ribosome fraction (Sucrose gradient separation) U937 cells DON Western Blotting 2 3 4 1 Monosome Polysome
    11. 11. 1 2 3 4 5 6 7 40s 60s 80s Fig. 4. P38 interacts with 40s ribosomal subunit and interaction is increased by DON-stimulated U937 cells. U937 cells (2 x 105/ml) were treated with DON (500 ng/ml) for 5 minutes compared with a vehicle. p38 pp38 RPS6 RPL7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 Vehicle DON
    12. 12. PKR and HCK mediate MAPK interaction with the ribosome. <ul><ul><li>PKR and HCK are important for MAPK interaction with the ribosome . </li></ul></ul><ul><ul><li>PKR can be upstream for HCK activation in the ribosome. </li></ul></ul><ul><ul><li>Ribosomal protein S3 can mediate MAPK interaction with the ribosome. </li></ul></ul>
    13. 13. p - p38 p38 2 - AP - - + + - - + + - - + + 15 min 30 min DON - - - - + + + + + + + + p - p38 p38 PP2 - - + + - - + + - - + + 15 min 30 min DON - - - - + + + + + + + + (A) (B)
    14. 14. pHCK p38 - 5 15 30 min DON Monosome(M) pp38 Ribosomal protein S3 can mediate MAPK interaction with the ribosome.
    15. 15. U9K-C2 HCK RPS6 (40S) PKR RPL7 (60S) 40s 60s 80s polysome U9K-A1 HCK PKR
    16. 16. Activated MAPK in the ribosome affect to transcription and translation. <ul><ul><li>Activated MAPK via the ribosome can move to cytoplasm and affect to polysome formation. </li></ul></ul><ul><ul><li>Activated MAPK can move to nucleus to change transcriptional level. </li></ul></ul>
    17. 17. P38 pP38 RPL7 P38 pP38 RPL7 - 5 15 30 min Polysome(P ) - 5 15 30 min Polysome(P ) Activated MAPK via the ribosome can affect polysome formation.
    18. 18. Cytoplasmic p38 MAP kinase can be moved to the perinuclear region by ribotoxic stress response. 1 2 3 4 5 1 2 3 4 5 p-p38 p38 p-p38 p38 A. B. RAW 264.7 cells were treated with DON 250ng/ml with different time point (1: vehicle, 2: 5min, 3: 15min, 4: 30min, 5: 60min) and lysed in PEB lysis buffer. After centrifugation at 10,000g for 15min, supernatents were collected and analyzed by western blot , A. The pellets were resuspensed into hot SDS lysis buffer with sonication, then centrifuged and analyzed by western blot , B. Calnexin Calnexin B.
    19. 19. Conformational change Activated kinases move to cytoplasm -> Signal pathway Polysome formation Selective mRNA Translation Gene Transcription mRNA Stabilization Ribosome Toxin Ribosome Activation of protein kinases PKR, HCK, ERK, JNK, p38

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