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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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(A) Nip3 mRNA of  1535 bp is shown with the open  reading frame boxed with the  predicted protein of 194 amino  acids. The putative transmembrane domain between amino  acids 164 and 184 of Nip3 protein is shaded. A mutant lacking  the transmembrane sequence was  constructed by truncation at  amino acid 163 and is called  Nip3163. (B) Nip3 Northern  blot of RNA extracted from  multiple murine tissues as indicated, and human MCF-7 mammary carcinoma cells. Transcript  sizes are noted on the right.
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Figure 1: (A) Nip3 mRNA of 1535 bp is shown with the open reading frame boxed with the predicted protein of 194 amino acids. The putative transmembrane domain between amino acids 164 and 184 of Nip3 protein is shaded. A mutant lacking the transmembrane sequence was constructed by truncation at amino acid 163 and is called Nip3163. (B) Nip3 Northern blot of RNA extracted from multiple murine tissues as indicated, and human MCF-7 mammary carcinoma cells. Transcript sizes are noted on the right.

Mentions: We isolated several Nip3 cDNAs from a human EBV transformed peripheral B lymphocyte library (Clonetech) by PCR and RACE of both 5′ and 3′ ends, and identified the longest as a 1,535-bp sequence (see Genebank accession number AF002697 for complete sequence). This is similar to the cDNA identified earlier (21) except in the 3′ UTR where we find no evidence of a homologous calbindin region in several independent experiments. The RNA has a long 3′ UTR and shorter 5′ UTR, and the encoded protein contains 194 amino acids and has a predicted molecular mass of ∼21.54 kD with a pI of 6.08. There is a putative transmembrane domain between amino acids 164 to 184 (Fig. 1 A). Nip3 mRNA is expressed in human breast carcinoma MCF-7 as a major transcript of ∼1.7 kb and two minor transcripts of 1.5 and 1.3 kb. The level of Nip3 mRNA in mouse tissues was also examined to determine how widely the gene is expressed. Two transcripts of 2.5 and 1.7-kb were identified with the 1.7-kb transcript found in all tissues examined and the larger transcript only in certain tissues such as brain, heart, kidney, liver, and submaxillary gland (Fig. 1 B). The identification of more than one Nip3 transcript in human and murine cells may indicate that the gene is alternately spliced or another closely related gene exists.


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)

(A) Nip3 mRNA of  1535 bp is shown with the open  reading frame boxed with the  predicted protein of 194 amino  acids. The putative transmembrane domain between amino  acids 164 and 184 of Nip3 protein is shaded. A mutant lacking  the transmembrane sequence was  constructed by truncation at  amino acid 163 and is called  Nip3163. (B) Nip3 Northern  blot of RNA extracted from  multiple murine tissues as indicated, and human MCF-7 mammary carcinoma cells. Transcript  sizes are noted on the right.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199165&req=5

Figure 1: (A) Nip3 mRNA of 1535 bp is shown with the open reading frame boxed with the predicted protein of 194 amino acids. The putative transmembrane domain between amino acids 164 and 184 of Nip3 protein is shaded. A mutant lacking the transmembrane sequence was constructed by truncation at amino acid 163 and is called Nip3163. (B) Nip3 Northern blot of RNA extracted from multiple murine tissues as indicated, and human MCF-7 mammary carcinoma cells. Transcript sizes are noted on the right.
Mentions: We isolated several Nip3 cDNAs from a human EBV transformed peripheral B lymphocyte library (Clonetech) by PCR and RACE of both 5′ and 3′ ends, and identified the longest as a 1,535-bp sequence (see Genebank accession number AF002697 for complete sequence). This is similar to the cDNA identified earlier (21) except in the 3′ UTR where we find no evidence of a homologous calbindin region in several independent experiments. The RNA has a long 3′ UTR and shorter 5′ UTR, and the encoded protein contains 194 amino acids and has a predicted molecular mass of ∼21.54 kD with a pI of 6.08. There is a putative transmembrane domain between amino acids 164 to 184 (Fig. 1 A). Nip3 mRNA is expressed in human breast carcinoma MCF-7 as a major transcript of ∼1.7 kb and two minor transcripts of 1.5 and 1.3 kb. The level of Nip3 mRNA in mouse tissues was also examined to determine how widely the gene is expressed. Two transcripts of 2.5 and 1.7-kb were identified with the 1.7-kb transcript found in all tissues examined and the larger transcript only in certain tissues such as brain, heart, kidney, liver, and submaxillary gland (Fig. 1 B). The identification of more than one Nip3 transcript in human and murine cells may indicate that the gene is alternately spliced or another closely related gene exists.

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