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Role for X-linked Inhibitor of apoptosis protein upstream of mitochondrial permeabilization.

Owens TW, Foster FM, Valentijn A, Gilmore AP, Streuli CH - J. Biol. Chem. (2009)

Bottom Line: Membrane-associated XIAP induces mitochondrial outer membrane permeabilization leading to cytochrome c and Smac release, which is dependent on Bax and Bak.We suggest that, as with Bcl-2 family proteins, more diverse functions for XIAP exist than previously identified.Moreover, switching the function of proteins from anti- to proapoptotic forms may be a common theme in the efficient execution of cell death.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom.

ABSTRACT
Apoptosis is controlled by a signaling equilibrium between prosurvival and proapoptotic pathways, such that unwanted apoptosis is avoided, but when required it occurs rapidly and efficiently. Many apoptosis regulators display dual roles, depending upon whether a cell has received an apoptotic stimulus or not. Here, we identify a novel and unexpected function for X-linked inhibitor of apoptosis (XIAP) that occurs when apoptosis is triggered under physiological conditions. We show that in response to loss of survival signals provided by cell adhesion, endogenous XIAP translocates from the cytosol into a mitochondrial 400-kDa complex and that this occurs very early in the apoptosis process. Membrane-associated XIAP induces mitochondrial outer membrane permeabilization leading to cytochrome c and Smac release, which is dependent on Bax and Bak. Thus, although XIAP suppresses apoptosis in healthy cells, our data indicate that XIAP may contribute to it in response to a proapoptotic signal such as loss of extracellular matrix-dependent survival signaling. We suggest that, as with Bcl-2 family proteins, more diverse functions for XIAP exist than previously identified. Moreover, switching the function of proteins from anti- to proapoptotic forms may be a common theme in the efficient execution of cell death.

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XIAP-induced cytochrome c release requires Bcl-2 family members. A, WT MEFs transiently expressing RFP or RFP-XIAP, or DKO MEFs expressing RFP-XIAP were fixed and scored for cytochrome c release. Note cytochrome c release in cells expressing RFP-XIAP (arrow), but mitochondrial cytochrome c in DKO cells expressing RFP-XIAP (arrowhead). B and C, DKO MEFs expressing RFP or RFP-XIAP only, or co-transfected with YFP or YFP-Bax, were scored for cytochrome c release (only transfected cells were counted). Representative images are shown in B; note that YFP-Bax is cytosolic in the RFP-expressing cell (arrowhead), whereas it has incorporated into clusters in the RFP-XIAP-expressing cell (arrow). D–F, WT MEFs and MECs expressing RFP-XIAP and either YFP-Bcl-XL or YFP-XT were scored for cytochrome c release. Representative images are shown in D.
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Figure 3: XIAP-induced cytochrome c release requires Bcl-2 family members. A, WT MEFs transiently expressing RFP or RFP-XIAP, or DKO MEFs expressing RFP-XIAP were fixed and scored for cytochrome c release. Note cytochrome c release in cells expressing RFP-XIAP (arrow), but mitochondrial cytochrome c in DKO cells expressing RFP-XIAP (arrowhead). B and C, DKO MEFs expressing RFP or RFP-XIAP only, or co-transfected with YFP or YFP-Bax, were scored for cytochrome c release (only transfected cells were counted). Representative images are shown in B; note that YFP-Bax is cytosolic in the RFP-expressing cell (arrowhead), whereas it has incorporated into clusters in the RFP-XIAP-expressing cell (arrow). D–F, WT MEFs and MECs expressing RFP-XIAP and either YFP-Bcl-XL or YFP-XT were scored for cytochrome c release. Representative images are shown in D.

Mentions: We asked whether the effect on MOMP was an intrinsic function of XIAP or whether it required the proapoptotic Bcl-2 family proteins, Bax and Bak. RFP or RFP-XIAP was expressed in either wild-type (WT) or DKO MEFs, and release of cytochrome c from mitochondria was examined (Fig. 3A). Although RFP-XIAP induced cytochrome c release in WT MEFs, it did not in DKO MEFs. To confirm this result, YFP-Bax was reexpressed in the DKO cells, together with RFP or RFP-XIAP (Fig. 3, B and C). Co-expression with YFP-Bax restored RFP-XIAP-induced cytochrome c release, whereas expression of YFP with RFP-XIAP did not. It is notable that in RFP-XIAP-expressing cells, YFP-Bax clustered into the large aggregates associated with MOMP (24, 25). Expression of YFP-Bak together with RFP-XIAP also rescued MOMP in DKO cells (data not shown).


Role for X-linked Inhibitor of apoptosis protein upstream of mitochondrial permeabilization.

Owens TW, Foster FM, Valentijn A, Gilmore AP, Streuli CH - J. Biol. Chem. (2009)

XIAP-induced cytochrome c release requires Bcl-2 family members. A, WT MEFs transiently expressing RFP or RFP-XIAP, or DKO MEFs expressing RFP-XIAP were fixed and scored for cytochrome c release. Note cytochrome c release in cells expressing RFP-XIAP (arrow), but mitochondrial cytochrome c in DKO cells expressing RFP-XIAP (arrowhead). B and C, DKO MEFs expressing RFP or RFP-XIAP only, or co-transfected with YFP or YFP-Bax, were scored for cytochrome c release (only transfected cells were counted). Representative images are shown in B; note that YFP-Bax is cytosolic in the RFP-expressing cell (arrowhead), whereas it has incorporated into clusters in the RFP-XIAP-expressing cell (arrow). D–F, WT MEFs and MECs expressing RFP-XIAP and either YFP-Bcl-XL or YFP-XT were scored for cytochrome c release. Representative images are shown in D.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 3: XIAP-induced cytochrome c release requires Bcl-2 family members. A, WT MEFs transiently expressing RFP or RFP-XIAP, or DKO MEFs expressing RFP-XIAP were fixed and scored for cytochrome c release. Note cytochrome c release in cells expressing RFP-XIAP (arrow), but mitochondrial cytochrome c in DKO cells expressing RFP-XIAP (arrowhead). B and C, DKO MEFs expressing RFP or RFP-XIAP only, or co-transfected with YFP or YFP-Bax, were scored for cytochrome c release (only transfected cells were counted). Representative images are shown in B; note that YFP-Bax is cytosolic in the RFP-expressing cell (arrowhead), whereas it has incorporated into clusters in the RFP-XIAP-expressing cell (arrow). D–F, WT MEFs and MECs expressing RFP-XIAP and either YFP-Bcl-XL or YFP-XT were scored for cytochrome c release. Representative images are shown in D.
Mentions: We asked whether the effect on MOMP was an intrinsic function of XIAP or whether it required the proapoptotic Bcl-2 family proteins, Bax and Bak. RFP or RFP-XIAP was expressed in either wild-type (WT) or DKO MEFs, and release of cytochrome c from mitochondria was examined (Fig. 3A). Although RFP-XIAP induced cytochrome c release in WT MEFs, it did not in DKO MEFs. To confirm this result, YFP-Bax was reexpressed in the DKO cells, together with RFP or RFP-XIAP (Fig. 3, B and C). Co-expression with YFP-Bax restored RFP-XIAP-induced cytochrome c release, whereas expression of YFP with RFP-XIAP did not. It is notable that in RFP-XIAP-expressing cells, YFP-Bax clustered into the large aggregates associated with MOMP (24, 25). Expression of YFP-Bak together with RFP-XIAP also rescued MOMP in DKO cells (data not shown).

Bottom Line: Membrane-associated XIAP induces mitochondrial outer membrane permeabilization leading to cytochrome c and Smac release, which is dependent on Bax and Bak.We suggest that, as with Bcl-2 family proteins, more diverse functions for XIAP exist than previously identified.Moreover, switching the function of proteins from anti- to proapoptotic forms may be a common theme in the efficient execution of cell death.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom.

ABSTRACT
Apoptosis is controlled by a signaling equilibrium between prosurvival and proapoptotic pathways, such that unwanted apoptosis is avoided, but when required it occurs rapidly and efficiently. Many apoptosis regulators display dual roles, depending upon whether a cell has received an apoptotic stimulus or not. Here, we identify a novel and unexpected function for X-linked inhibitor of apoptosis (XIAP) that occurs when apoptosis is triggered under physiological conditions. We show that in response to loss of survival signals provided by cell adhesion, endogenous XIAP translocates from the cytosol into a mitochondrial 400-kDa complex and that this occurs very early in the apoptosis process. Membrane-associated XIAP induces mitochondrial outer membrane permeabilization leading to cytochrome c and Smac release, which is dependent on Bax and Bak. Thus, although XIAP suppresses apoptosis in healthy cells, our data indicate that XIAP may contribute to it in response to a proapoptotic signal such as loss of extracellular matrix-dependent survival signaling. We suggest that, as with Bcl-2 family proteins, more diverse functions for XIAP exist than previously identified. Moreover, switching the function of proteins from anti- to proapoptotic forms may be a common theme in the efficient execution of cell death.

Show MeSH
Related in: MedlinePlus