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Apoptosis induction by Bid requires unconventional ubiquitination and degradation of its N-terminal fragment.

Tait SW, de Vries E, Maas C, Keller AM, D'Santos CS, Borst J - J. Cell Biol. (2007)

Bottom Line: We found that, upon Bid cleavage, the N-terminal fragment (tBid-N) is ubiquitinated and degraded, thus freeing the BH3 domain in the C-terminal fragment (tBid-C).Ubiquitination of tBid-N is unconventional because acceptor sites are neither lysines nor the N terminus.We conclude that unconventional ubiquitination and proteasome-dependent degradation of tBid-N is required to unleash the proapoptotic activity of tBid-C.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, The Netherlands Cancer Institute, 1066 CX Amsterdam, Netherlands.

ABSTRACT
Bcl-2 family member Bid is subject to autoinhibition; in the absence of stimuli, its N-terminal region sequesters the proapoptotic Bcl-2 homology 3 (BH3) domain. Upon proteolytic cleavage in its unstructured loop, Bid is activated, although structural data reveal no apparent resulting conformational change. We found that, upon Bid cleavage, the N-terminal fragment (tBid-N) is ubiquitinated and degraded, thus freeing the BH3 domain in the C-terminal fragment (tBid-C). Ubiquitination of tBid-N is unconventional because acceptor sites are neither lysines nor the N terminus. Chemical approaches implicated thioester and hydroxyester linkage of ubiquitin and mutagenesis implicated serine and possibly threonine as acceptor residues in addition to cysteine. Acceptor sites reside predominantly but not exclusively in helix 1, which is required for ubiquitination and degradation of tBid-N. Rescue of tBid-N from degradation blocked Bid's ability to induce mitochondrial outer membrane permeability but not mitochondrial translocation of the cleaved complex. We conclude that unconventional ubiquitination and proteasome-dependent degradation of tBid-N is required to unleash the proapoptotic activity of tBid-C.

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Related in: MedlinePlus

Stabilization of tBid-N prohibits tBid-C function. (A) MCF-7 cells were depleted of endogenous Bid by stable expression of a Bid siRNA. MCF-7 Bid RNAi cells were reconstituted by transient transfection with Myc Bid GFP or GFP Bid VSV with silent mutations to allow escape from RNAi. The results for reconstitution with Myc Bid GFP wild-type and RNAi escape mutants are shown by immunoblotting for GFP in total cell lysates. Molecular masses (kD) of marker proteins are indicated. (B) Myc Bid GFP and GFP Bid VSV were expressed in MCF-7 Bid RNAi cells and cells were stimulated for the indicated time periods with TNF-α. Full-length Bid and cleavage fragments were detected by anti-Bid immunoblotting of total cell lysates. Fragments were identified additionally by immunoblotting with antibodies to Myc, GFP, and VSV (not depicted). ECL signals originate from the same blotted gel but the bottom panel represents a longer exposure than the top to allow detection of Myc tBid-N. (C) MCF-7 cells expressing GFP Bid VSV were stimulated with TNF-α for 8 h or left untreated (control). Signals diagnostic for stabilized GFP tBid-N are shown individually and in combination with the signals for tBid-C (VSV) and MitoTracker. The enlarged views allow assessment of colocalization of tBid-N and tBid-C at mitochondria. (D) TNF-α–induced Cyt c release was tested after 8 h of stimulation by flow cytometric analysis in parental MCF-7 cells, cells lacking Bid (MCF-7 Bid RNAi), and the same cells reconstituted with either Myc Bid GFP (unstable tBid-N) or GFP Bid VSV (stabilized GFP tBid-N). MCF-7 and MCF-7 Bid RNAi cells were additionally transfected to express histone-GFP. Cyt c content was monitored in cells gated for equal GFP expression to standardize for transfected Bid levels. Results are means + SD from three independent experiments. Values are corrected for Cyt c release in the absence of stimulus (15 ± 5%). The asterisk indicates statistically significant difference (P < 0.05 according to a t test).
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fig7: Stabilization of tBid-N prohibits tBid-C function. (A) MCF-7 cells were depleted of endogenous Bid by stable expression of a Bid siRNA. MCF-7 Bid RNAi cells were reconstituted by transient transfection with Myc Bid GFP or GFP Bid VSV with silent mutations to allow escape from RNAi. The results for reconstitution with Myc Bid GFP wild-type and RNAi escape mutants are shown by immunoblotting for GFP in total cell lysates. Molecular masses (kD) of marker proteins are indicated. (B) Myc Bid GFP and GFP Bid VSV were expressed in MCF-7 Bid RNAi cells and cells were stimulated for the indicated time periods with TNF-α. Full-length Bid and cleavage fragments were detected by anti-Bid immunoblotting of total cell lysates. Fragments were identified additionally by immunoblotting with antibodies to Myc, GFP, and VSV (not depicted). ECL signals originate from the same blotted gel but the bottom panel represents a longer exposure than the top to allow detection of Myc tBid-N. (C) MCF-7 cells expressing GFP Bid VSV were stimulated with TNF-α for 8 h or left untreated (control). Signals diagnostic for stabilized GFP tBid-N are shown individually and in combination with the signals for tBid-C (VSV) and MitoTracker. The enlarged views allow assessment of colocalization of tBid-N and tBid-C at mitochondria. (D) TNF-α–induced Cyt c release was tested after 8 h of stimulation by flow cytometric analysis in parental MCF-7 cells, cells lacking Bid (MCF-7 Bid RNAi), and the same cells reconstituted with either Myc Bid GFP (unstable tBid-N) or GFP Bid VSV (stabilized GFP tBid-N). MCF-7 and MCF-7 Bid RNAi cells were additionally transfected to express histone-GFP. Cyt c content was monitored in cells gated for equal GFP expression to standardize for transfected Bid levels. Results are means + SD from three independent experiments. Values are corrected for Cyt c release in the absence of stimulus (15 ± 5%). The asterisk indicates statistically significant difference (P < 0.05 according to a t test).

Mentions: To create a cell line that exclusively expressed stabilized tBid-N, endogenous Bid was constitutively down-regulated in MCF-7 cells by RNAi (Fig. 7 A). To express stabilized GFP Bid VSV and control (unstable) Myc Bid GFP in these cells, a silent mutation was introduced into the Bid sequence that was not targeted by the siRNA. This allowed appropriate expression of the Bid constructs upon transient transfection (Fig. 7, A and B). The processing of Myc Bid GFP and GFP Bid VSV by caspase-8/10 and the half-life of the Bid fragments were followed kinetically after stimulation of the reconstituted MCF-7 cells with TNF-α. In the case of Myc Bid GFP, the tBid-C GFP cleavage product was visible from the 2-h time point onward and its signal increased in time. The Myc tBid-N fragment was detected at the 2- and 4-h time points but not at the 8-h time point (Fig. 7 B). Processing of GFP Bid VSV was also visible at 2 h, but in this case not only the tBid-C VSV fragment remained detectable in the 8-h time frame but also the GFP tBid-N fragment (Fig. 7 B). We conclude that the GFP Bid VSV protein is properly processed by caspase-8/10 and that the GFP tBid-N fragment is long-lived, whereas the Myc tBid-N fragment has a short half-life.


Apoptosis induction by Bid requires unconventional ubiquitination and degradation of its N-terminal fragment.

Tait SW, de Vries E, Maas C, Keller AM, D'Santos CS, Borst J - J. Cell Biol. (2007)

Stabilization of tBid-N prohibits tBid-C function. (A) MCF-7 cells were depleted of endogenous Bid by stable expression of a Bid siRNA. MCF-7 Bid RNAi cells were reconstituted by transient transfection with Myc Bid GFP or GFP Bid VSV with silent mutations to allow escape from RNAi. The results for reconstitution with Myc Bid GFP wild-type and RNAi escape mutants are shown by immunoblotting for GFP in total cell lysates. Molecular masses (kD) of marker proteins are indicated. (B) Myc Bid GFP and GFP Bid VSV were expressed in MCF-7 Bid RNAi cells and cells were stimulated for the indicated time periods with TNF-α. Full-length Bid and cleavage fragments were detected by anti-Bid immunoblotting of total cell lysates. Fragments were identified additionally by immunoblotting with antibodies to Myc, GFP, and VSV (not depicted). ECL signals originate from the same blotted gel but the bottom panel represents a longer exposure than the top to allow detection of Myc tBid-N. (C) MCF-7 cells expressing GFP Bid VSV were stimulated with TNF-α for 8 h or left untreated (control). Signals diagnostic for stabilized GFP tBid-N are shown individually and in combination with the signals for tBid-C (VSV) and MitoTracker. The enlarged views allow assessment of colocalization of tBid-N and tBid-C at mitochondria. (D) TNF-α–induced Cyt c release was tested after 8 h of stimulation by flow cytometric analysis in parental MCF-7 cells, cells lacking Bid (MCF-7 Bid RNAi), and the same cells reconstituted with either Myc Bid GFP (unstable tBid-N) or GFP Bid VSV (stabilized GFP tBid-N). MCF-7 and MCF-7 Bid RNAi cells were additionally transfected to express histone-GFP. Cyt c content was monitored in cells gated for equal GFP expression to standardize for transfected Bid levels. Results are means + SD from three independent experiments. Values are corrected for Cyt c release in the absence of stimulus (15 ± 5%). The asterisk indicates statistically significant difference (P < 0.05 according to a t test).
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fig7: Stabilization of tBid-N prohibits tBid-C function. (A) MCF-7 cells were depleted of endogenous Bid by stable expression of a Bid siRNA. MCF-7 Bid RNAi cells were reconstituted by transient transfection with Myc Bid GFP or GFP Bid VSV with silent mutations to allow escape from RNAi. The results for reconstitution with Myc Bid GFP wild-type and RNAi escape mutants are shown by immunoblotting for GFP in total cell lysates. Molecular masses (kD) of marker proteins are indicated. (B) Myc Bid GFP and GFP Bid VSV were expressed in MCF-7 Bid RNAi cells and cells were stimulated for the indicated time periods with TNF-α. Full-length Bid and cleavage fragments were detected by anti-Bid immunoblotting of total cell lysates. Fragments were identified additionally by immunoblotting with antibodies to Myc, GFP, and VSV (not depicted). ECL signals originate from the same blotted gel but the bottom panel represents a longer exposure than the top to allow detection of Myc tBid-N. (C) MCF-7 cells expressing GFP Bid VSV were stimulated with TNF-α for 8 h or left untreated (control). Signals diagnostic for stabilized GFP tBid-N are shown individually and in combination with the signals for tBid-C (VSV) and MitoTracker. The enlarged views allow assessment of colocalization of tBid-N and tBid-C at mitochondria. (D) TNF-α–induced Cyt c release was tested after 8 h of stimulation by flow cytometric analysis in parental MCF-7 cells, cells lacking Bid (MCF-7 Bid RNAi), and the same cells reconstituted with either Myc Bid GFP (unstable tBid-N) or GFP Bid VSV (stabilized GFP tBid-N). MCF-7 and MCF-7 Bid RNAi cells were additionally transfected to express histone-GFP. Cyt c content was monitored in cells gated for equal GFP expression to standardize for transfected Bid levels. Results are means + SD from three independent experiments. Values are corrected for Cyt c release in the absence of stimulus (15 ± 5%). The asterisk indicates statistically significant difference (P < 0.05 according to a t test).
Mentions: To create a cell line that exclusively expressed stabilized tBid-N, endogenous Bid was constitutively down-regulated in MCF-7 cells by RNAi (Fig. 7 A). To express stabilized GFP Bid VSV and control (unstable) Myc Bid GFP in these cells, a silent mutation was introduced into the Bid sequence that was not targeted by the siRNA. This allowed appropriate expression of the Bid constructs upon transient transfection (Fig. 7, A and B). The processing of Myc Bid GFP and GFP Bid VSV by caspase-8/10 and the half-life of the Bid fragments were followed kinetically after stimulation of the reconstituted MCF-7 cells with TNF-α. In the case of Myc Bid GFP, the tBid-C GFP cleavage product was visible from the 2-h time point onward and its signal increased in time. The Myc tBid-N fragment was detected at the 2- and 4-h time points but not at the 8-h time point (Fig. 7 B). Processing of GFP Bid VSV was also visible at 2 h, but in this case not only the tBid-C VSV fragment remained detectable in the 8-h time frame but also the GFP tBid-N fragment (Fig. 7 B). We conclude that the GFP Bid VSV protein is properly processed by caspase-8/10 and that the GFP tBid-N fragment is long-lived, whereas the Myc tBid-N fragment has a short half-life.

Bottom Line: We found that, upon Bid cleavage, the N-terminal fragment (tBid-N) is ubiquitinated and degraded, thus freeing the BH3 domain in the C-terminal fragment (tBid-C).Ubiquitination of tBid-N is unconventional because acceptor sites are neither lysines nor the N terminus.We conclude that unconventional ubiquitination and proteasome-dependent degradation of tBid-N is required to unleash the proapoptotic activity of tBid-C.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, The Netherlands Cancer Institute, 1066 CX Amsterdam, Netherlands.

ABSTRACT
Bcl-2 family member Bid is subject to autoinhibition; in the absence of stimuli, its N-terminal region sequesters the proapoptotic Bcl-2 homology 3 (BH3) domain. Upon proteolytic cleavage in its unstructured loop, Bid is activated, although structural data reveal no apparent resulting conformational change. We found that, upon Bid cleavage, the N-terminal fragment (tBid-N) is ubiquitinated and degraded, thus freeing the BH3 domain in the C-terminal fragment (tBid-C). Ubiquitination of tBid-N is unconventional because acceptor sites are neither lysines nor the N terminus. Chemical approaches implicated thioester and hydroxyester linkage of ubiquitin and mutagenesis implicated serine and possibly threonine as acceptor residues in addition to cysteine. Acceptor sites reside predominantly but not exclusively in helix 1, which is required for ubiquitination and degradation of tBid-N. Rescue of tBid-N from degradation blocked Bid's ability to induce mitochondrial outer membrane permeability but not mitochondrial translocation of the cleaved complex. We conclude that unconventional ubiquitination and proteasome-dependent degradation of tBid-N is required to unleash the proapoptotic activity of tBid-C.

Show MeSH
Related in: MedlinePlus