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The E1B 19K/Bcl-2-binding protein Nip3 is a dimeric mitochondrial protein that activates apoptosis.

Chen G, Ray R, Dubik D, Shi L, Cizeau J, Bleackley RC, Saxena S, Gietz RD, Greenberg AH - J. Exp. Med. (1997)

Bottom Line: Nip3 is expressed in mitochondria and a mutant (Nip3(163)) lacking the putative transmembrane domain and COOH terminus does not dimerize or localize to mitochondria.After transfection, both Nip3 and Nip3(163) protein levels decrease steadily over 48 h indicating that the protein is rapidly degraded and this occurs in the absence of cell death.In conclusion, Nip3 is an apoptosis-inducing dimeric mitochondrial protein that can overcome Bcl-2 suppression.

View Article: PubMed Central - PubMed

Affiliation: Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba, Canada.

ABSTRACT
Nip3 (nineteen kD interacting protein-3) is an E1B 19K and Bcl-2 binding protein of unknown function. Nip3 is detected as both a 60- and 30-kD protein in vivo and in vitro and exhibits strong homologous interaction in a yeast two-hybrid system indicating that it can homodimerize. Nip3 is expressed in mitochondria and a mutant (Nip3(163)) lacking the putative transmembrane domain and COOH terminus does not dimerize or localize to mitochondria. Transient transfection of epitope-tagged Nip3 in Rat-1 fibroblasts and MCF-7 breast carcinoma induces apoptosis within 12 h while cells transfected with the Nip3(163) mutant have a normal phenotype, suggesting that mitochondrial localization is necessary for induction of cell death. Nip3 overexpression increases the sensitivity to apoptosis induced by granzyme B and topoisomerase I and II inhibitors. After transfection, both Nip3 and Nip3(163) protein levels decrease steadily over 48 h indicating that the protein is rapidly degraded and this occurs in the absence of cell death. Bcl-2 overexpression initially delays the onset of apoptosis induced by Nip3 but the resistance is completely overcome in longer periods of incubation. Nip3 protein levels are much higher and persist longer in Bcl-2 expressing cells. In conclusion, Nip3 is an apoptosis-inducing dimeric mitochondrial protein that can overcome Bcl-2 suppression.

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Nip3 overcomes Bcl-2 suppression of apoptosis. (A) (Left)  Rat-1 and Rat-1/Bcl-2 cells were transfected with T7-Nip3 and at increasing time the cells were harvested and Nip3-expressing apoptotic  cells quantitated as described in Fig. 4. (Right) The Rat-1 and Rat-1/ Bcl-2 cells were treated with granzyme B as shown at a constant concentration of perforin (125 ng/ml) for 3 h and apoptotic cells quantitated by Hoechst dye as described previously (22). Three other experiments showed similar results. (B) After transfection of Rat-1 and Rat-1/ Bcl-2 cells with T7-Nip3, lysates were harvested at increasing time intervals then Western blotted with anti-T7 antibody. Molecular mass  markers are shown on the left. (C) Western blot of Bcl-2 (arrow) in  Rat-1 (lane 1) and Rat-1/Bcl-2 (lane 2) cell lines. Blots were developed with rabbit anti–human Bcl-2 (PharMingen). Relative molecular  mass (kD) is shown on the left.
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Figure 6: Nip3 overcomes Bcl-2 suppression of apoptosis. (A) (Left) Rat-1 and Rat-1/Bcl-2 cells were transfected with T7-Nip3 and at increasing time the cells were harvested and Nip3-expressing apoptotic cells quantitated as described in Fig. 4. (Right) The Rat-1 and Rat-1/ Bcl-2 cells were treated with granzyme B as shown at a constant concentration of perforin (125 ng/ml) for 3 h and apoptotic cells quantitated by Hoechst dye as described previously (22). Three other experiments showed similar results. (B) After transfection of Rat-1 and Rat-1/ Bcl-2 cells with T7-Nip3, lysates were harvested at increasing time intervals then Western blotted with anti-T7 antibody. Molecular mass markers are shown on the left. (C) Western blot of Bcl-2 (arrow) in Rat-1 (lane 1) and Rat-1/Bcl-2 (lane 2) cell lines. Blots were developed with rabbit anti–human Bcl-2 (PharMingen). Relative molecular mass (kD) is shown on the left.

Mentions: Since it had been demonstrated that Bcl-2 binds Nip3 (21), we next determined if Bcl-2 was capable of suppressing Nip3-induced apoptosis. Rat-1 and Rat-1/Bcl-2 cells (Fig. 6 C) were transfected with T7-Nip3 and apoptotic cells appearing in both populations was determined over time. Apoptotic Rat-1 cells expressing Nip3 were detected within 12 h and reached maximum levels by 48 h, while Nip3 did not induce cell death in Rat-1/Bcl-2 cells until 24 h after transfection. However, by 48 h virtually all cells expressing Nip3 were apoptotic despite Bcl-2 overexpression (Fig. 6 A). The Rat-1 cells expressing Bcl-2 were highly resistant to granzyme B and perforin (Fig. 6 A). We noticed that Nip3 protein level was considerably higher in Rat-1/Bcl-2 cells than in Rat-1 cells by 12 h after transfection and remained higher throughout the time course. However, a significant decrease in Nip3 level was detected by 48–60 h in Rat-1/ Bcl-2 cells at the same time that recovery of apoptosis was detected (Fig. 6 B).


The E1B 19K/Bcl-2-binding protein Nip3 is a dimeric mitochondrial protein that activates apoptosis.

Chen G, Ray R, Dubik D, Shi L, Cizeau J, Bleackley RC, Saxena S, Gietz RD, Greenberg AH - J. Exp. Med. (1997)

Nip3 overcomes Bcl-2 suppression of apoptosis. (A) (Left)  Rat-1 and Rat-1/Bcl-2 cells were transfected with T7-Nip3 and at increasing time the cells were harvested and Nip3-expressing apoptotic  cells quantitated as described in Fig. 4. (Right) The Rat-1 and Rat-1/ Bcl-2 cells were treated with granzyme B as shown at a constant concentration of perforin (125 ng/ml) for 3 h and apoptotic cells quantitated by Hoechst dye as described previously (22). Three other experiments showed similar results. (B) After transfection of Rat-1 and Rat-1/ Bcl-2 cells with T7-Nip3, lysates were harvested at increasing time intervals then Western blotted with anti-T7 antibody. Molecular mass  markers are shown on the left. (C) Western blot of Bcl-2 (arrow) in  Rat-1 (lane 1) and Rat-1/Bcl-2 (lane 2) cell lines. Blots were developed with rabbit anti–human Bcl-2 (PharMingen). Relative molecular  mass (kD) is shown on the left.
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Figure 6: Nip3 overcomes Bcl-2 suppression of apoptosis. (A) (Left) Rat-1 and Rat-1/Bcl-2 cells were transfected with T7-Nip3 and at increasing time the cells were harvested and Nip3-expressing apoptotic cells quantitated as described in Fig. 4. (Right) The Rat-1 and Rat-1/ Bcl-2 cells were treated with granzyme B as shown at a constant concentration of perforin (125 ng/ml) for 3 h and apoptotic cells quantitated by Hoechst dye as described previously (22). Three other experiments showed similar results. (B) After transfection of Rat-1 and Rat-1/ Bcl-2 cells with T7-Nip3, lysates were harvested at increasing time intervals then Western blotted with anti-T7 antibody. Molecular mass markers are shown on the left. (C) Western blot of Bcl-2 (arrow) in Rat-1 (lane 1) and Rat-1/Bcl-2 (lane 2) cell lines. Blots were developed with rabbit anti–human Bcl-2 (PharMingen). Relative molecular mass (kD) is shown on the left.
Mentions: Since it had been demonstrated that Bcl-2 binds Nip3 (21), we next determined if Bcl-2 was capable of suppressing Nip3-induced apoptosis. Rat-1 and Rat-1/Bcl-2 cells (Fig. 6 C) were transfected with T7-Nip3 and apoptotic cells appearing in both populations was determined over time. Apoptotic Rat-1 cells expressing Nip3 were detected within 12 h and reached maximum levels by 48 h, while Nip3 did not induce cell death in Rat-1/Bcl-2 cells until 24 h after transfection. However, by 48 h virtually all cells expressing Nip3 were apoptotic despite Bcl-2 overexpression (Fig. 6 A). The Rat-1 cells expressing Bcl-2 were highly resistant to granzyme B and perforin (Fig. 6 A). We noticed that Nip3 protein level was considerably higher in Rat-1/Bcl-2 cells than in Rat-1 cells by 12 h after transfection and remained higher throughout the time course. However, a significant decrease in Nip3 level was detected by 48–60 h in Rat-1/ Bcl-2 cells at the same time that recovery of apoptosis was detected (Fig. 6 B).

Bottom Line: Nip3 is expressed in mitochondria and a mutant (Nip3(163)) lacking the putative transmembrane domain and COOH terminus does not dimerize or localize to mitochondria.After transfection, both Nip3 and Nip3(163) protein levels decrease steadily over 48 h indicating that the protein is rapidly degraded and this occurs in the absence of cell death.In conclusion, Nip3 is an apoptosis-inducing dimeric mitochondrial protein that can overcome Bcl-2 suppression.

View Article: PubMed Central - PubMed

Affiliation: Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba, Canada.

ABSTRACT
Nip3 (nineteen kD interacting protein-3) is an E1B 19K and Bcl-2 binding protein of unknown function. Nip3 is detected as both a 60- and 30-kD protein in vivo and in vitro and exhibits strong homologous interaction in a yeast two-hybrid system indicating that it can homodimerize. Nip3 is expressed in mitochondria and a mutant (Nip3(163)) lacking the putative transmembrane domain and COOH terminus does not dimerize or localize to mitochondria. Transient transfection of epitope-tagged Nip3 in Rat-1 fibroblasts and MCF-7 breast carcinoma induces apoptosis within 12 h while cells transfected with the Nip3(163) mutant have a normal phenotype, suggesting that mitochondrial localization is necessary for induction of cell death. Nip3 overexpression increases the sensitivity to apoptosis induced by granzyme B and topoisomerase I and II inhibitors. After transfection, both Nip3 and Nip3(163) protein levels decrease steadily over 48 h indicating that the protein is rapidly degraded and this occurs in the absence of cell death. Bcl-2 overexpression initially delays the onset of apoptosis induced by Nip3 but the resistance is completely overcome in longer periods of incubation. Nip3 protein levels are much higher and persist longer in Bcl-2 expressing cells. In conclusion, Nip3 is an apoptosis-inducing dimeric mitochondrial protein that can overcome Bcl-2 suppression.

Show MeSH
Related in: MedlinePlus