Pro-apoptotic Proteins Released from the Mitochondria Regulate the Protein Composition and Caspase-processing Activity of the Native Apaf-1/Caspase-9 Apoptosome Complex

摘要

The apoptosome is a large caspase-activating (∼700–1400 kDa) complex, which is assembled from Apaf-1 and caspase-9 when cytochrome c is released during mitochondrial-dependent apoptotic cell death. Apaf-1 the core scaffold protein is ∼135 kDa and contains CARD (caspase recruitment domain), CED-4, and multiple (13Benedict M.A. Hu Y. Inohara N. Nunez G. J. Biol. Chem. 2000; 275: 8461-8468Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) WD40 repeat domains, which can potentially interact with a variety of unknown regulatory proteins. To identify such proteins we activated THP.1 lysates with dATP/cytochrome c and used sucrose density centrifugation and affinity-based methods to purify the apoptosome for analysis by MALDI-TOF mass spectrometry. First, we used a glutathione S-transferase (GST) fusion protein (GST-casp91–130) containing the CARD domain of caspase-9-(1–130), which binds to the CARD domain of Apaf-1 when it is in the apoptosome and blocks recruitment/activation of caspase-9. This affinity-purified apoptosome complex contained only Apaf-1XL and GST-casp91–130, demonstrating that the WD40 and CED-4 domains of Apaf-1 do proteins. we used a to caspase-9 to the apoptosome complex from cell containing of cytochrome of This apoptosome complex and contained of of of and cytochrome which only to the in lysates containing and the of the apoptosome complex and contained only Apaf-1 and the of caspase-9. and cytochrome c released from and it is that the apoptosome complex in apoptotic of Apaf-1 and caspase-9. The apoptosome is a large caspase-activating (∼700–1400 kDa) complex, which is assembled from Apaf-1 and caspase-9 when cytochrome c is released during mitochondrial-dependent apoptotic cell death. Apaf-1 the core scaffold protein is ∼135 kDa and contains CARD (caspase recruitment domain), CED-4, and multiple (13Benedict M.A. Hu Y. Inohara N. Nunez G. J. Biol. Chem. 2000; 275: 8461-8468Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) WD40 repeat domains, which can potentially interact with a variety of unknown regulatory proteins. To identify such proteins we activated THP.1 lysates with dATP/cytochrome c and used sucrose density centrifugation and affinity-based methods to purify the apoptosome for analysis by MALDI-TOF mass spectrometry. First, we used a glutathione S-transferase (GST) fusion protein (GST-casp91–130) containing the CARD domain of caspase-9-(1–130), which binds to the CARD domain of Apaf-1 when it is in the apoptosome and blocks recruitment/activation of caspase-9. This affinity-purified apoptosome complex contained only Apaf-1XL and GST-casp91–130, demonstrating that the WD40 and CED-4 domains of Apaf-1 do proteins. we used a to caspase-9 to the apoptosome complex from cell containing of cytochrome of This apoptosome complex and contained of of of and cytochrome which only to the in lysates containing and the of the apoptosome complex and contained only Apaf-1 and the of caspase-9. and cytochrome c released from and it is that the apoptosome complex in apoptotic of Apaf-1 and caspase-9. The and of apoptotic cell from the of a of J. PubMed Scopus Google PubMed Google PubMed Scopus Google The of is a of and of from to and to and which for the of apoptotic cell death. CED-4 is to and cell by Full Text Full Text PDF PubMed Scopus Google of CED-4, which and and of Y. 2000; PubMed Scopus Google for a which is to in of which in cell in which in activated in a that a which in the and and of cell of 2000; PubMed Scopus Google The is to in that it the of the a large complex Full Text Full Text PDF PubMed Scopus Google PubMed Scopus Google of and by Apaf-1 a of CED-4 only by a in cell lysates by and apoptotic J. Full Text Full Text PDF PubMed Scopus Google Apaf-1 a CED-4 Full Text Full Text PDF PubMed Scopus Google cytochrome c J. Full Text Full Text PDF PubMed Scopus Google Scholar) and caspase-9 Full Text Full Text PDF PubMed Scopus Google Apaf-1 is a large ∼135 kDa which of r