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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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In vitro expression of Nip3 as a homodimer. (A) After in vitro transcription and translation of Nip3 the 35S-labeled protein was separated on  SDS-PAGE (lanes 1 and 2) under reducing (R, lane 1) and nonreducing (NR, lane 2) conditions. Nip3 runs as two bands at 60 and 30 kD. Nip3163 is a  truncated mutant in which the terminal 31 amino acids from 164 to 194 containing the putative transmembrane domain have been removed (lanes 3 and  4). The truncated Nip3163 is expressed as a major band at 29 kD and a minor band at 28 kD under both reducing (lane 3) and nonreducing (lane 4) conditions. (B) Comparative peptide mapping of in vitro translated 35S-labeled 60-kD Nip3 and 28-kD Nip3163 protein. After in vitro translation, Nip3 and  Nip3163 were immunoprecipitated, the 60- and 28-kD bands recovered after SDS-PAGE then trypsin digested. The resulting peptides were separated on  the same plate by electrophoresis at pH 1.9 (horizontal dimension with anode to the left), followed by ascending chromatography. The positions of the  origin of Nip3 and Nip3163 are marked by arrows. Three [35S]methionine-labeled peptides predicted from the trypsin digest are circled and labeled A, B,  and C (Nip3) and A′, B′, and C′ (Nip3163). Peptide A and A′, amino acids 1–45; B and B′, amino acids 140–146; C and C′, 147–152. All three peptides  are represented in both Nip3 and Nip3163. Minor spots are similar in the two proteins and likely are partially digested fragments. One of two experiments  with similar results is shown. (C) Yeast two-hybrid system identifies Nip3 homodimerization. (left) Plasmids encoding full-length Nip3 and the COOH-terminal truncated mutant Nip3163 fused to the GAL4 DNA-binding domain were cotransformed with plasmids encoding Nip3, Nip3163 or empty vector sequences fused to the GAL4 transcriptional activation domain. Protein–protein interactions were determined by growth of yeast in the absence of leucine,  tryptophan and histidine. (Right) Growth in the absence of leucine and tryptophan is shown as a control. The results are representative of 3 independent  experiments.
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Figure 2: In vitro expression of Nip3 as a homodimer. (A) After in vitro transcription and translation of Nip3 the 35S-labeled protein was separated on SDS-PAGE (lanes 1 and 2) under reducing (R, lane 1) and nonreducing (NR, lane 2) conditions. Nip3 runs as two bands at 60 and 30 kD. Nip3163 is a truncated mutant in which the terminal 31 amino acids from 164 to 194 containing the putative transmembrane domain have been removed (lanes 3 and 4). The truncated Nip3163 is expressed as a major band at 29 kD and a minor band at 28 kD under both reducing (lane 3) and nonreducing (lane 4) conditions. (B) Comparative peptide mapping of in vitro translated 35S-labeled 60-kD Nip3 and 28-kD Nip3163 protein. After in vitro translation, Nip3 and Nip3163 were immunoprecipitated, the 60- and 28-kD bands recovered after SDS-PAGE then trypsin digested. The resulting peptides were separated on the same plate by electrophoresis at pH 1.9 (horizontal dimension with anode to the left), followed by ascending chromatography. The positions of the origin of Nip3 and Nip3163 are marked by arrows. Three [35S]methionine-labeled peptides predicted from the trypsin digest are circled and labeled A, B, and C (Nip3) and A′, B′, and C′ (Nip3163). Peptide A and A′, amino acids 1–45; B and B′, amino acids 140–146; C and C′, 147–152. All three peptides are represented in both Nip3 and Nip3163. Minor spots are similar in the two proteins and likely are partially digested fragments. One of two experiments with similar results is shown. (C) Yeast two-hybrid system identifies Nip3 homodimerization. (left) Plasmids encoding full-length Nip3 and the COOH-terminal truncated mutant Nip3163 fused to the GAL4 DNA-binding domain were cotransformed with plasmids encoding Nip3, Nip3163 or empty vector sequences fused to the GAL4 transcriptional activation domain. Protein–protein interactions were determined by growth of yeast in the absence of leucine, tryptophan and histidine. (Right) Growth in the absence of leucine and tryptophan is shown as a control. The results are representative of 3 independent experiments.

Mentions: We prepared [35S]methionine-labeled Nip3 by in vitro transcription and translation and examined the protein on SDS-PAGE. Nip3 was seen as a major band at ∼60 kD and a smaller band of 30 kD (Fig. 2 A). Nip3 has a predicted molecular mass of ∼21.54 kD, much smaller than the size of the two bands estimated from SDS-PAGE, indicating that the protein runs anomalously. The two bands were seen under both reducing and nonreducing conditions. However, when the COOH terminus from amino acid 163 was removed (Nip3163) (Fig. 1 A), the large molecular mass form was absent and a major band of 28 kD and a minor band of 27 kD were detected. These data suggest that the 30-kD protein is a Nip3 monomer while the 60-kD protein is a dimer that was formed by interaction at the COOH terminus of the protein. To examine more directly if Nip3 is a homodimer, we compared tryptic peptide fragments of the 60-kD Nip3 to the 28-kD Nip3163. After in vitro translation, [35S]methionine-labeled Nip3 and Nip3163 were purified by immunoprecipitation with mouse mAb to Nip3 and SDS PAGE. Tryptic peptides were prepared and fragments separated by 2D electrophoresis and chromatography and detected by radioautography as described in Materials and Methods. Three peptide fragments of Nip3 and Nip3163 contain methionine, while the COOH-terminal fragments including the truncated region of Nip3163 do not. Fig. 2 B illustrates that the two proteins have three major [35S]methionine-labeled peptides and their positions are very similar after 2D mapping. No other major peptides are detected in the 60-kD Nip3 band. Minor proteins are also similar and likely represent partially digested Nip3.


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)

In vitro expression of Nip3 as a homodimer. (A) After in vitro transcription and translation of Nip3 the 35S-labeled protein was separated on  SDS-PAGE (lanes 1 and 2) under reducing (R, lane 1) and nonreducing (NR, lane 2) conditions. Nip3 runs as two bands at 60 and 30 kD. Nip3163 is a  truncated mutant in which the terminal 31 amino acids from 164 to 194 containing the putative transmembrane domain have been removed (lanes 3 and  4). The truncated Nip3163 is expressed as a major band at 29 kD and a minor band at 28 kD under both reducing (lane 3) and nonreducing (lane 4) conditions. (B) Comparative peptide mapping of in vitro translated 35S-labeled 60-kD Nip3 and 28-kD Nip3163 protein. After in vitro translation, Nip3 and  Nip3163 were immunoprecipitated, the 60- and 28-kD bands recovered after SDS-PAGE then trypsin digested. The resulting peptides were separated on  the same plate by electrophoresis at pH 1.9 (horizontal dimension with anode to the left), followed by ascending chromatography. The positions of the  origin of Nip3 and Nip3163 are marked by arrows. Three [35S]methionine-labeled peptides predicted from the trypsin digest are circled and labeled A, B,  and C (Nip3) and A′, B′, and C′ (Nip3163). Peptide A and A′, amino acids 1–45; B and B′, amino acids 140–146; C and C′, 147–152. All three peptides  are represented in both Nip3 and Nip3163. Minor spots are similar in the two proteins and likely are partially digested fragments. One of two experiments  with similar results is shown. (C) Yeast two-hybrid system identifies Nip3 homodimerization. (left) Plasmids encoding full-length Nip3 and the COOH-terminal truncated mutant Nip3163 fused to the GAL4 DNA-binding domain were cotransformed with plasmids encoding Nip3, Nip3163 or empty vector sequences fused to the GAL4 transcriptional activation domain. Protein–protein interactions were determined by growth of yeast in the absence of leucine,  tryptophan and histidine. (Right) Growth in the absence of leucine and tryptophan is shown as a control. The results are representative of 3 independent  experiments.
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Figure 2: In vitro expression of Nip3 as a homodimer. (A) After in vitro transcription and translation of Nip3 the 35S-labeled protein was separated on SDS-PAGE (lanes 1 and 2) under reducing (R, lane 1) and nonreducing (NR, lane 2) conditions. Nip3 runs as two bands at 60 and 30 kD. Nip3163 is a truncated mutant in which the terminal 31 amino acids from 164 to 194 containing the putative transmembrane domain have been removed (lanes 3 and 4). The truncated Nip3163 is expressed as a major band at 29 kD and a minor band at 28 kD under both reducing (lane 3) and nonreducing (lane 4) conditions. (B) Comparative peptide mapping of in vitro translated 35S-labeled 60-kD Nip3 and 28-kD Nip3163 protein. After in vitro translation, Nip3 and Nip3163 were immunoprecipitated, the 60- and 28-kD bands recovered after SDS-PAGE then trypsin digested. The resulting peptides were separated on the same plate by electrophoresis at pH 1.9 (horizontal dimension with anode to the left), followed by ascending chromatography. The positions of the origin of Nip3 and Nip3163 are marked by arrows. Three [35S]methionine-labeled peptides predicted from the trypsin digest are circled and labeled A, B, and C (Nip3) and A′, B′, and C′ (Nip3163). Peptide A and A′, amino acids 1–45; B and B′, amino acids 140–146; C and C′, 147–152. All three peptides are represented in both Nip3 and Nip3163. Minor spots are similar in the two proteins and likely are partially digested fragments. One of two experiments with similar results is shown. (C) Yeast two-hybrid system identifies Nip3 homodimerization. (left) Plasmids encoding full-length Nip3 and the COOH-terminal truncated mutant Nip3163 fused to the GAL4 DNA-binding domain were cotransformed with plasmids encoding Nip3, Nip3163 or empty vector sequences fused to the GAL4 transcriptional activation domain. Protein–protein interactions were determined by growth of yeast in the absence of leucine, tryptophan and histidine. (Right) Growth in the absence of leucine and tryptophan is shown as a control. The results are representative of 3 independent experiments.
Mentions: We prepared [35S]methionine-labeled Nip3 by in vitro transcription and translation and examined the protein on SDS-PAGE. Nip3 was seen as a major band at ∼60 kD and a smaller band of 30 kD (Fig. 2 A). Nip3 has a predicted molecular mass of ∼21.54 kD, much smaller than the size of the two bands estimated from SDS-PAGE, indicating that the protein runs anomalously. The two bands were seen under both reducing and nonreducing conditions. However, when the COOH terminus from amino acid 163 was removed (Nip3163) (Fig. 1 A), the large molecular mass form was absent and a major band of 28 kD and a minor band of 27 kD were detected. These data suggest that the 30-kD protein is a Nip3 monomer while the 60-kD protein is a dimer that was formed by interaction at the COOH terminus of the protein. To examine more directly if Nip3 is a homodimer, we compared tryptic peptide fragments of the 60-kD Nip3 to the 28-kD Nip3163. After in vitro translation, [35S]methionine-labeled Nip3 and Nip3163 were purified by immunoprecipitation with mouse mAb to Nip3 and SDS PAGE. Tryptic peptides were prepared and fragments separated by 2D electrophoresis and chromatography and detected by radioautography as described in Materials and Methods. Three peptide fragments of Nip3 and Nip3163 contain methionine, while the COOH-terminal fragments including the truncated region of Nip3163 do not. Fig. 2 B illustrates that the two proteins have three major [35S]methionine-labeled peptides and their positions are very similar after 2D mapping. No other major peptides are detected in the 60-kD Nip3 band. Minor proteins are also similar and likely represent partially digested Nip3.

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