From the Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba R3E 0V9, Canada
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ABSTRACT |
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We have identified Nix, a homolog of the E1B
19K/Bcl-2 binding and pro-apoptotic protein Nip3. Human and murine Nix
have a 56 and 53% amino acid identity to human and murine Nip3,
respectively. The carboxyl terminus of Nix, including a transmembrane
domain, is highly homologous to Nip3 but it bears a longer and distinct asparagine/proline-rich N terminus. Human Nip3 maps to chromosome 14q11.2-q12, whereas Nix/BNip3L was found on 8q21. Nix encodes a
23.8-kDa protein but it is expressed as a 48-kDa protein, suggesting that it homodimerizes similarly to Nip3. Following transfection, Nix
protein undergoes progressive proteolysis to an 11-kDa C-terminal fragment, which is blocked by the proteasome inhibitor lactacystin. Nix
colocalizes with the mitochondrial matrix protein HSP60, and removal of
the putative transmembrane domain (TM) results in general cytoplasmic
and nuclear expression. When transiently expressed, Nix and Nip3 but
not TM deletion mutants rapidly activate apoptosis. Nix can overcome
the suppressers Bcl-2 and Bcl-XL, although high levels of Bcl-XL expression will inhibit apoptosis. We
propose that Nix and Nip3 form a new subfamily of pro-apoptotic
mitochondrial proteins.
Apoptosis is regulated by molecules that both activate and
suppress cell death. The molecular regulators of developmental apoptosis in Caenorhabditis elegans include cell death
inducers Ced-3 and -4 (1) which are conserved in mammals (2, 3). Ced-9
is the prototypical cell death suppresser protein, and Bcl-2 and
Bcl-XL are the mammalian homologs (4). However, the Bcl-2 family of proteins also contains members that function to activate apoptosis such as Bax, Bad, Bak, Bik/Nbk, and Hrk (5-8).
We recently identified a new cell death-inducing protein named Nip3 (9)
(also called BNip3 (10)), which was initially reported to physically
interact with both adenovirus E1B 19K and Bcl-2 (11). Nip3 is a
mitochondrial protein that induces apoptosis and can enhance the
apoptosis induced by other cell death signals when transiently
expressed (9, 10). Bcl-2 transiently suppresses Nip3-induced apoptosis
although this resistance is rapidly overcome (9, 10). We report the
cloning of Nix (Nip3-like protein X), which
closely resembles Nip3 in both structure and function. We propose that
Nix and Nip3 form a new structurally related pro-apoptotic family of
mitochondrial proteins.
Cloning of Murine Nix (mNix), Human Nix (hNix), and Murine Nip3
(mNip3)--
Searches of the GenBank
EST1 and TIGR (Institute for
Genomic Research) data bank using the human Nip3 cDNA revealed
related but novel human and mouse EST clones. Using primers within
these EST sequences, we generated PCR fragments, which were cloned into pCRII vector (Invitrogen, San Diego, CA) and sequenced. The entire mNix
and hNix cDNA were then polymerase chain reacted from mouse 17-day
embryo Matchmaker cDNA library and human fetal liver 5'-stretch plus cDNA library (CLONTECH, Palo Alto, CA).
Murine Nip3 cDNA was cloned from a Reagents and Cell Lines--
Rat-1, Rat-1/Bcl-2, TC (10T1/2
parental cell line), TX5 and TX22 (Bcl-XL transfected
clones), and MCF-7 cells were culture in Northern Analysis--
Total RNA (30 µg) was fractionated on a
formaldehyde denaturing gel and then transferred and hybridized with
nick-translated human or mouse Nix cDNA corresponding to the coding
region as described (9).
In Vitro Transcription--
35S-labeled Nix proteins
were prepared by in vitro transcription and translation of
cDNA cloned in pcDNA3 vector using TnT coupled reticulocyte
lysate system (Promega, Madison, WI).
Western Blotting--
Aliquots of 5 × 104
cells transfected with Nix-T7 were separated by an SDS-PAGE method
using Tris-Tricine buffer and polyvinylidene difluoride membrane that
is suitable for detecting small molecular weight peptides (13). Western
blots were probed with mouse monoclonal anti-T7 antibody using an ECL
detection kit (Amersham Pharmacia Biotech, UK).
Transient Transfection and Apoptosis Assays--
Cells were
transfected with Nix using LipofectAMINE Reagent (Life Technologies,
Inc.). Aliquots of 5 × 104 cells were Western blotted
as described above. For apoptosis experiments, Nix-expressing cells
were detected with mouse anti-T7 antibody and FITC-conjugated rabbit
anti-mouse IgG antibody. Apoptotic cells, exhibiting altered DNA
morphology following Hoechst dye staining, were enumerated manually
counting as described previously (9). Fluorescence was visualized using
a Zeiss Axiophot microscope equipped with a cooled CCD camera.
For The cloning of hNix, mNix, and mNip3 was as follows. Using the
human Nip3 cDNA as a probe, we screened a
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MATERIALS AND METHODS
GT11 3T3 fibroblast cDNA
library using human Nip3 as a probe. KpnI and
EcoRI sites were introduced into the full-length cDNA by
PCR and then subcloned into expression vector pcDNA3-T7, a modified
pcDNA3 vector containing a C-terminal T7-tag (9). Transmembrane
domain-deleted expression vector mNix (
TM) was generated by PCR
using splice overlap extension (12). Human Nip3 and the
Nip3163 transmembrane deletion mutant have been described
previously (9).
-minimal essential medium
(Life Technologies, Inc.) supplemented with 10% fetal bovine serum
(Life Technologies, Inc.). Murine monoclonal anti-T7 antibody was
purchased from Novagen (Madison, WI). Rabbit anti-HSP60 antibody was
donated by Dr. Radhey Gupta (McMaster University). FITC-conjugated goat
anti-rabbit IgG (Sigma Chemical Co.) and Cy3-conjugated goat anti-mouse
IgG (BioCan Ltd., Mississauga) were used for fluorescence studies.
Lactacystin was purchased from Calbiochem-Novabiochem (Cambridge, MA).
-galactosidase apoptosis assays evaluating the effect of
removing the transmembrane domain on Nix and Nip3 apoptosis, 293T cells
were cotransfected with 0.2 µg of pcDNA3-
-galactosidase plus
0.75 µg of the expression plasmid in triplicate using LipofectAMINE. Cells were fixed, stained, and evaluated 21 h later as described previously (14).
RESULTS AND DISCUSSION
GT11 mouse fibroblast cDNA library for clones related to Nip3. The clone containing the
longest insert of 1311 bp was found to contain the entire mouse Nip3
coding region of 187 amino acids (GenBank accession number AF041054).
The cDNA had overall identity with human Nip3 of 90.2% (Fig.
1).
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Fig. 1.
Alignment of predicted murine and human Nip3
and Nix proteins. Regions of identity with hNix are
shaded. The predicted transmembrane domains are
underlined.
Human and murine ESTs related to but different from Nip3 were used for
PCR to clone, as described under "Materials and Methods," a human
1337-bp and murine 1351-bp cDNA containing a large open reading
frame of 219 and 218 amino acids, respectively. The protein was named
Nix. The predicted human Nix (hNix) (GenBank accession number AF067396)
and mouse Nix (mNix) (AF067395) proteins share 56 and 53% identity,
respectively, with Nip3 of the same species, and are 97% identical to
each other (Fig. 1). The highest regions of homology between the four
proteins resides in the C terminus. Nix proteins bear a single
hydrophobic -helical sequence at the extreme carboxyl terminus,
corresponding to the location of the TM domain of Nip3 (2) (Fig. 1).
Nix has a significantly longer N terminus that contains an unusual five
asparagines in sequence and is proline-rich. N-terminal amino acids
1-59 of hNix and 1-32 of hNip3 are the most divergent. Another region
from amino acids 97-120 of hNix is also distinct from hNip3, whereas the remainder is over 85% homologous with large blocks of identical sequences in both species.
Similar to Nip3 (9, 11), Nix contains PEST sequences comprised of high frequency stretches of Pro, Glu, Ser, Thr, and Asp, flanked by charged amino acids His, Arg, or Lys. PEST sequences are associated with proteins having high turnover rates (15) whose degradation is controlled by the proteasome. hNix and mNix are expressed as 4.5- and 1.4-kilobase mRNA transcripts, respectively (not shown). The smaller transcript corresponds to the size of the cDNAs reported above and is likely the complete sequence. The larger transcript has not been identified; however, some cDNA clones were found that bear a distinct 5'-UTR suggesting alternate splicing of the Nix mRNA, and may represent a larger transcript.
Following in vitro transcription and translation and Western blotting (Fig. 2A), mNix and hNix run as proteins of 48,000 relative molecular weight under reducing and non-reducing conditions using the Tris-Tricine SDS-PAGE method (13). hNip3 has a 40,000 relative molecular weight in Tris-Tricine-buffered SDS-PAGE (Fig. 2A). In contrast, using the Laemmli method, Nix runs as an 80-kDa protein (not shown) and Nip3 at 60 kDa (9). The reason for the difference in mobility in the two buffers is not clear; however Nip3, which forms a homodimer that does not dissociate under reducing conditions (9), runs at its predicted molecular weight of 40,000 in the Tris-Tricine SDS-PAGE. Nix is also unaffected by reduction and is about double its 23.8 kDa predicted mass (Fig. 2A), suggesting that it may also be a homodimer.
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Western blotting of lysates from Rat-1 cells transiently transfected with expression vector Nix-T7 reveals a 48-kDa protein that is progressively degraded over 42 h (Fig. 2B). We examined both N- and C-terminal T7-tagged Nix and found that a residual 11-kDa fragment was detected only with the C-terminal tag (Fig. 2B). Nix with an N-terminal tag showed a similar degradation pattern of the 48-kDa band but no smaller fragments were detected (not shown). Intermediate fragments were also visible on some transfections at earlier time points or after prolonged development of the blots but only with the C-terminal tag. A similar pattern of degradation was found following transfection with Nip3 in which an 11-kDa fragment was detected using a C- but not an N-terminal tag. The 11-kDa residual Nip3 fragment was only detectable using the Tris-Tricine-buffered SDS-PAGE, which is optimized for the detection of small peptides. The proteolytic degradation is dependent on an active proteasome. Nix remains as a 48-kDa protein when cells were first treated with lactacystin, the proteasome threonine protease inhibitor (Fig. 2B). Nip3 is also degraded rapidly following transfection (9), and as with Nix, Nip3 degradation is also inhibited in lactacystin-treated cells (not shown).
To determine whether Nix is localized to mitochondria, the primary site
of Nip3 expression (9, 10), mNix-T7 was transiently transfected into
Rat-1 cells and stained with both monoclonal anti-T7 and rabbit
anti-HSP60 antibodies. Secondary antibodies, FITC-labeled goat
anti-mouse IgG and Cy3-labeled goat anti-rabbit IgG, were then used to
visualize the localization of Nix and HSP60, respectively, within the
cells after immunofluorescence and confocal microscopy. HSP60 is
primarily located in mitochondria (16), and our results showed that Nix
colocalizes with HSP60 (Fig.
3A). Because Nip3 requires the
TM domain to localize to mitochondria, we prepared a mutant Nix without
the putative TM domain (Nix TM). Nix
TM is expressed evenly
throughout the cytoplasm and is not localized to any organelle-like
structure (Fig. 3B). Unlike Nip3
TM (9), the Nix mutant
was detected in the nucleus in higher levels than in the cytoplasm.
Because Nip3 induces apoptosis when transiently transfected, we
examined the effects of Nix expression in Rat-1 and MCF-7 cells over
time. Nix-expressing cells undergo apoptosis (Fig. 3C). This
was quantitated over a 48-h period, and a progressive increase in the
percent of cells that were apoptotic was observed (Fig.
4A) and at about the same rate as that in
Nip3 (Fig. 4B). Removal of the transmembrane domain of Nix
prevented the induction of cell death similar to that of transmembrane
deficient mutant of Nip3 (Fig. 4C).
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Nip3-induced apoptosis is only partially suppressed in Bcl-2 expressing cell lines (9, 10). Inhibition is detected early in the time course but is overcome at later time points (9). Examining the apoptosis rate over a 48-h period following mNix transfection, we found that there was no significant difference between Rat-1 and Rat-1/Bcl-2 cell lines (Fig. 5A), the latter being highly resistant to other apoptotic signals such as granzyme B (9). At decreasing doses of Nix plasmid from 1.0 to 0.1 µg, the transfection efficiency decreased progressively but the apoptosis rate in transfected cells remained constant between the two cell lines (not shown). Examining Nix apoptosis in Bcl-XL expressing cell lines TX22 and TX5, we find that only the higher Bcl-XL expressing cell line TX5 was effective at blocking Nix although both lines were resistant to staurosporine-induced apoptosis when compared with the parental line TC (Fig. 5,B and C).
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The suppressive action of Bcl-2 can be reversed by heterodimerization with pro-apoptotic members of the Bcl-2 family through the BH3 (Bcl-2 homology-3) domain (8, 17, 18). Nip3 protein appears to have a region that bears some structural similarity to the BH3 domain of the Bcl-2 family (9, 10), which may partially function to promote Nip3 cell death (10). However, it is also clear that Nip3 and Nix are more structurally related to each other than to pro-apoptotic members of the Bcl-2 family.
A recent report identified a human cDNA called BNip3L that is homologous to hNip3 and maps to chromosome 8p21 (19). The protein encoded by the BNip3L cDNA is identical to the human Nix. For comparison, we have now mapped the chromosomal location of human Nip3 and, using a radiation hybrid panel, we find it is on chromosome 14; 4.08 centi-Ray from the marker WI-6506. Using the Whitehead Institute/MIT YAC map of chromosome 14, the CEPH YAC 951d6 was positive, placing the gene slightly centromeric to WI-6506 at the band 14q11.2-q12. The distinct mapping location of the two genes warrants a unique name for Nix rather than BNip3L because Nix is not a long form of Nip3.
Expression of BNip3L in cervical cancer cell lines reduced their colony-forming ability and suggested to the authors that the protein may have growth-suppressive function. Because we find that both hNix and mNix are pro-apoptotic when overexpressed in tumor cell lines, induction of apoptosis can account for the observed growth suppression and poor colony formation in the BNip3L-transfected carcinoma cells.
In conclusion, we have described Nix, a homolog of the Nip3 protein,
which shares the ability to induce apoptosis, localize to mitochondria,
and undergo proteasome-dependent degradation when
overexpressed. Nip3 and Nix form a new subfamily of mitochondrial cell
death proteins.
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ACKNOWLEDGEMENTS |
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We thank Dr. Brian Fristensky for analyzing the Nix and Nip3 proteins and Dr. Radhey Gupta for the anti-HSP60 antibody. We also thank the Medical Research Council Genome Resource Facility at The Hospital for Sick Children, Toronto, for radiation and YAC mapping.
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FOOTNOTES |
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* This work was supported by the National Cancer Institute of Canada and Medical Research Council of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF041054, AF067396, and AF067395.
Terry Fox Cancer Research Scientist. To whom correspondence should
be addressed: 100 Olivia St., Manitoba Institute of Cell Biology,
University of Manitoba, Winnipeg, MB R3E 0V9, Canada. Tel.:
204-787-2112; Fax: 204-787-2190; E-mail:
agreenb{at}cc.umanitoba.ca.
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ABBREVIATIONS |
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The abbreviations used are: EST, expressed sequence tag; PCR, polymerase chain reaction; TM, transmembrane; FITC, fluorescein isothiocyanate; PAGE, polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; bp, base pair(s)..
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