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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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Overexpression of Nip3 but not Nip3163 induces apoptosis.  (A) Rat-1 or MCF-7 (left) cells were transfected with T7-tagged Nip3 then  at different times cells harvested and stained with anti-T7 antibody and  FITC anti–mouse IgG antibody to identify cells expressing Nip3. The  frequency of apoptotic cells was quantitated by Hoechst dye staining.  Rat-1 cells (right) were then transfected and apoptotic cells expressing (•)  and not expressing (○) Nip3 were quantitated. Rat-1 cells expressing (▾)  or not expressing (▿) Nip3163 were analysed in the same manner. At least  200 cells were counted in each sample. All assays were repeated three to  seven times with identical results. (B) Western blot of Rat-1 cells transfected with T7-Nip3 (left) or T7-Nip3163 (right). Cells were harvested at  the times indicated and Western blots developed with anti-T7 antibody.
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Figure 4: Overexpression of Nip3 but not Nip3163 induces apoptosis. (A) Rat-1 or MCF-7 (left) cells were transfected with T7-tagged Nip3 then at different times cells harvested and stained with anti-T7 antibody and FITC anti–mouse IgG antibody to identify cells expressing Nip3. The frequency of apoptotic cells was quantitated by Hoechst dye staining. Rat-1 cells (right) were then transfected and apoptotic cells expressing (•) and not expressing (○) Nip3 were quantitated. Rat-1 cells expressing (▾) or not expressing (▿) Nip3163 were analysed in the same manner. At least 200 cells were counted in each sample. All assays were repeated three to seven times with identical results. (B) Western blot of Rat-1 cells transfected with T7-Nip3 (left) or T7-Nip3163 (right). Cells were harvested at the times indicated and Western blots developed with anti-T7 antibody.

Mentions: T7 epitope-tagged Nip3 was transiently expressed in Rat-1 or MCF-7 cells. Cells were stained with both anti-T7 antibody to identify cells expressing Nip3, and Hoechst dye to quantitate apoptosis. Over time, progressively more Nip3 expressing cells became apoptotic starting around 12 h and reaching completion by 48 to 60 h (Fig. 4 A). Nip3 protein measured by anti-T7 Western blot were highest between 12 and 24 h after transfection then progressively decreased with time paralleling the induction of apoptosis (Fig. 4 B). To determine if the transmembrane domain of Nip3 which is necessary for mitochondrial localization was also necessary to induce apoptosis, the experiment was repeated using the Nip3163. The mutant was unable to induce apoptosis (Fig. 4, A and B). Of additional interest, Nip3163 protein levels decreased very rapidly with time despite the inability of the mutant to induce apoptosis, thus indicating that the loss of protein in the cell lysates was not related to the death of cells.


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)

Overexpression of Nip3 but not Nip3163 induces apoptosis.  (A) Rat-1 or MCF-7 (left) cells were transfected with T7-tagged Nip3 then  at different times cells harvested and stained with anti-T7 antibody and  FITC anti–mouse IgG antibody to identify cells expressing Nip3. The  frequency of apoptotic cells was quantitated by Hoechst dye staining.  Rat-1 cells (right) were then transfected and apoptotic cells expressing (•)  and not expressing (○) Nip3 were quantitated. Rat-1 cells expressing (▾)  or not expressing (▿) Nip3163 were analysed in the same manner. At least  200 cells were counted in each sample. All assays were repeated three to  seven times with identical results. (B) Western blot of Rat-1 cells transfected with T7-Nip3 (left) or T7-Nip3163 (right). Cells were harvested at  the times indicated and Western blots developed with anti-T7 antibody.
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Related In: Results  -  Collection

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Figure 4: Overexpression of Nip3 but not Nip3163 induces apoptosis. (A) Rat-1 or MCF-7 (left) cells were transfected with T7-tagged Nip3 then at different times cells harvested and stained with anti-T7 antibody and FITC anti–mouse IgG antibody to identify cells expressing Nip3. The frequency of apoptotic cells was quantitated by Hoechst dye staining. Rat-1 cells (right) were then transfected and apoptotic cells expressing (•) and not expressing (○) Nip3 were quantitated. Rat-1 cells expressing (▾) or not expressing (▿) Nip3163 were analysed in the same manner. At least 200 cells were counted in each sample. All assays were repeated three to seven times with identical results. (B) Western blot of Rat-1 cells transfected with T7-Nip3 (left) or T7-Nip3163 (right). Cells were harvested at the times indicated and Western blots developed with anti-T7 antibody.
Mentions: T7 epitope-tagged Nip3 was transiently expressed in Rat-1 or MCF-7 cells. Cells were stained with both anti-T7 antibody to identify cells expressing Nip3, and Hoechst dye to quantitate apoptosis. Over time, progressively more Nip3 expressing cells became apoptotic starting around 12 h and reaching completion by 48 to 60 h (Fig. 4 A). Nip3 protein measured by anti-T7 Western blot were highest between 12 and 24 h after transfection then progressively decreased with time paralleling the induction of apoptosis (Fig. 4 B). To determine if the transmembrane domain of Nip3 which is necessary for mitochondrial localization was also necessary to induce apoptosis, the experiment was repeated using the Nip3163. The mutant was unable to induce apoptosis (Fig. 4, A and B). Of additional interest, Nip3163 protein levels decreased very rapidly with time despite the inability of the mutant to induce apoptosis, thus indicating that the loss of protein in the cell lysates was not related to the death of cells.

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