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Cleavage of IP 3 R1 by caspase-3 and intracellular Ca 2+ homeostasis during Apoptosis

Cleavage of IP 3 R1 by caspase-3 and intracellular Ca 2+ homeostasis during Apoptosis. Laboratory of Physiology, KULeuven, Belgium Assefa Z, Bultynck G, Szlufcik K, Nadif Kasri N, Vermassen E, Goris J, Missiaen L, Callewaert G, Parys JB & De Smedt H. Apoptosis: brief introduction.

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Cleavage of IP 3 R1 by caspase-3 and intracellular Ca 2+ homeostasis during Apoptosis

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  1. Cleavage of IP3R1 by caspase-3 and intracellular Ca2+ homeostasis during Apoptosis Laboratory of Physiology, KULeuven, Belgium Assefa Z, Bultynck G, Szlufcik K, Nadif Kasri N, Vermassen E, Goris J, Missiaen L, Callewaert G, Parys JB & De Smedt H

  2. Apoptosis: brief introduction • genetically controlled form of cell death critical for normal embryonic development and adult tissue homeostasis. • dysregulation of apoptosis is associated with a number of disease conditions. • characterized by: • - PS exposure • - cytoplasmic and nuclear shrinkage • - cell membrane blebbing • - chromatic condensation • - intranucleosomal DNA fragmentation • - Fragmentation into membrane-bound apoptotic bodies • involves caspase activity.

  3. Apoptosis: various ways of dying Ca2+ cardiolipin calpain calcineurin endonucleases caspase FADD Pro-caspase-8 Apaf-1 caspase-6 caspase-7 caspase-6 caspase-7

  4. Apoptosis: the role of IP3Rs . are directly implicated in apoptosis: * Expression of antisense cDNA construct of IP3R3 blocks dexamethasone-induced apoptosis and increase in [Ca2+]i in lymphocytes. Khan et al.,Science 273: 503, 1996 * IP3R1 deficient cells were resistant to Fas, dexamethasone and g-irradiation. Jayaraman et al.,Mol. Cell Biol. 17: 3005, 1997 * BCR-induced apoptosis in lymphocytes was significantly inhibited only in cells deficient of all three receptors. Sugawara et al.,EMBO J. 16: 3078, 1997 * IP3R1 is a substrate of caspase-3 during apoptosis. Hirota et al.,JBC 274: 34433, 1999

  5.  1-225 Caspase-3 cleavage Cytochrome C binding 31 25 13 18 CaM FKBP12

  6. IP3R1 constructs: cytoplasm N C ER lumen N C N C - the constructs were stably expressed in DT40 cells that lack all three IP3Rs.

  7. Del(1-225)-IP3R1 is no longer functional

  8. Localization of (D1-1891)IP3R1 expressed in IP3R triple ko cells (D1-1891)IP3R1-Mitotracker (D1-1891)IP3R1 -bodipy TG

  9. Cleavage of IP3R1 invitro and in cells undergoing apoptosis

  10. Caspase-3 mediated cleavage of IP3R1leads to an increase in ... Caspase-3 activity Apoptosis

  11. Secondary necrosis in STS-treated cells

  12. Apoptosis-related rise in [Ca2+]i requires caspase cleavage of IP3R1. Fluo-3 Fura red WT-IP3R1 Fluo-3 Fura red IP3R1Dcasp (D1-225)IP3R1 IP3R-KO fluorescence intensity (relative units) (D1-1891)IP3R1 fluorescence intensity (relative units)

  13. Caspase-3 activation precedes the increase in [Ca2+]i WT-IP3R1

  14. Caspase inhibitors block the rise in [Ca2+ ]i WT-IP3R1 Fluo-3 Fura red (D1-225)IP3R1 fluorescence intensity

  15. Apoptotic cell death and the associated rise in [Ca2+]i could be induced in Ca2+-free medium WT-IP3R1 (D1-225)IP3R1 (D1-1891)IP3R1 Ca2+-free Normal

  16. (D1-1891)IP3R1 WT-IP3R1 (D1-1891)IP3R1 expression does not reduce the amount of releasable Ca2+ from the ER

  17. Staurosporine Anti-chicken IgM [Ca2+]i Caspase-3activation IP3R1cleavage IP3-independentactivity Apoptosis Role of IP3R1 in apoptotic cell death

  18. CONCLUSIONS: • 1. Calcium release through IP3R1 is required for the rapid execution of apoptotic cell death; but that doesn’t involve IICR activity but cleavage by caspase-3. • 2. The disruption of intracellular Ca2+ homeostasis during apoptosis seems to be the consequence rather than the primary cause of apoptosis. • 3. The specific pattern of changes in [Ca2+]i during apoptosis in different cell types might be related to the relative distribution of IP3R1 among the different tissues.

  19. CaBP CaM -Ca2+ +Ca2+ RACK1 Gβ AKAP9 PP1 PKA PP1 REGULATORY SITES Ca2+-binding sitesPKA/PKG phosphoryl.ATP binding Caspase-3 cleavage N IP3 C Type 1 IP3R (IP3R1)

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