Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M405747200 on July 15, 2004

J. Biol. Chem., Vol. 279, Issue 38, 39942-39950, September 17, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/38/39942    most recent
M405747200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cho, D.-H.
Right arrow Articles by Jung, Y.-K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cho, D.-H.
Right arrow Articles by Jung, Y.-K.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Induced Inhibition of Ischemic/Hypoxic Injury by APIP, a Novel Apaf-1-interacting Protein*

Dong-Hyung Cho{ddagger}§, Yeon-Mi Hong{ddagger}, Ho-June Lee{ddagger}, Ha-Na Woo{ddagger}, Jong-Ok Pyo{ddagger}, Tak W. Mak¶, and Yong-Keun Jung{ddagger}||

From the {ddagger}Department of Life Science, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea and the Department of Cellular and Molecular Biology, Ontario Cancer Institute, Toronto, Ontario M5G 2C1, Canada

Received for publication, May 24, 2004 , and in revised form, July 15, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We describe the isolation and characterization of a new apaf-1-interacting protein (APIP) as a negative regulator of ischemic injury. APIP is highly expressed in skeletal muscle and heart and binds to the CARD of Apaf-1 in competition with caspase-9. Exogenous APIP inhibits cytochrome c-induced activation of caspase-3 and caspase-9, and suppresses cell death triggered by mitochondrial apoptotic stimuli through inhibiting the downstream activity of cytochrome c released from mitochondria. Conversely, reduction of APIP expression potentiates mitochondrial apoptosis. APIP expression is highly induced in mouse muscle affected by ischemia produced by interruption of the artery in the hindlimb and in C2C12 myotubes created by hypoxia in vitro, and the blockade of APIP up-regulation results in TUNEL-positive ischemic damage. Furthermore, forced expression of APIP suppresses ischemia/hypoxia-induced death of skeletal muscle cells. Taken together, these results suggest that APIP functions to inhibit muscle ischemic damage by binding to Apaf-1 in the Apaf-1/caspase-9 apoptosis pathway.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Apaf-1 has been identified as the mammalian homolog of Caenorhabditis elegans ced-4, which is a core component of the cell death machine (together with ced-3 and ced-4) and plays a crucial role in the general apoptotic program of normal development and pathogenesis (1-4). Understanding the pathways that lead to apoptosis and identifying strategies to regulate this pathway may have important clinical implications. In stress-induced apoptosis, mitochondria releases apoptogenic factors such as cytochrome c, AIF, Smac/Diablo, and serine protease HtrA2 (5-10). Pro-apoptotic members of the Bcl-2 family such as Bax and Bid induce the release of apoptogenic factors, whereas anti-apoptotic members such as Bcl-2 or Bcl-XL prevent their releases (11). Bid is cleaved by caspase-8 and the truncated Bid (tBid) translocates to the mitochondria, where it triggers Bax activation (12-14). Cytochrome c binds to Apaf-1 and, when dATP is added to the cytochrome c-bound Apaf-1, the oligomeric complex of Apaf-1 forms and leads to the recruitment of caspase-9 to form apoptosome and activates caspase-9 (15-17).

Previous reports on Apaf-1 and caspase-9 knockout mice suggest that Apaf-1 is involved in the control of cell numbers in the developing brain, retina, face, and limbs. Apaf-1 and caspase-9 are essential for caspase-3 activation and seem to play important roles in normal development (18-20). Apaf-1-deficient mice exhibit reduced apoptosis in the brain and striking craniofacial abnormalities with hyperproliferation of neuronal cells. The thymocytes from Apaf-1 knockout mice fail to undergo apoptosis after treatment with various apoptotic stimuli. Apaf-1 and caspase-9 are also reported to control tumor development by functionally interacting with p53 (21). Although some apoptosis regulated by Bcl-2 is activated independently of the apoptosome (22, 23), these observations indicate that Apaf-1 plays a central role in the common events of mitochondria-dependent cell death pathways. Therefore, identification of such molecules as Boo and Hsp70 as regulators of Apaf-1-mediated cell death will be important (24-26).

Ischemia and reperfusion of skeletal muscle occurs in acute vascular occlusion and revascularization, in elective vascular surgery, in orthopedic surgery by means of a tourniquet, and in transplantation of muscle containing cutaneous flaps. Such ischemic injury to skeletal muscle induces free oxygen radical production, a loss of energy supply to the cell, a loss of cellular calcium homeostasis, or activation of apoptosis pathway (27, 28). However, the control circuit to regulate ischemic damage to skeletal muscle is largely unknown.

In this study, we isolated and characterized the Apaf-1-interacting protein, APIP.1 APIP is highly expressed in skeletal muscle and its expression regulates Apaf-1-mediated cell death. In particular, APIP expression is induced during ischemia as a necessary step leading to the suppression of hypoxia-induced muscle cell death.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Yeast Two-hybrid Assay—The CARD region (amino acids 1-89) of Apaf-1 was used as bait in conventional yeast two-hybrid screening using pLexA vector (Clontech, Palo Alto, CA) and Jurkat cDNA library (Stratagene, La Jolla, CA).

Plasmid Construction—The CARD of Apaf-1 was amplified by PCR and subcloned into the BamH' site of pLexA (pLexA-CARD). The mouse CARD of Apaf-1 was subcloned into BamH' and Xho' site of pcDNA-FLAG (pmCARD-FLAG). cDNAs encoding the full open reading frames of APIP and APIP2 were subcloned into the Xho' site of pcDNA3-HA (pHA-APIP) and pEGFP (pGFP-APIP). Antisense cDNAs of APIP and FADD were subcloned into pcDNA3-HA (pAS-APIP) and pEGFP (pAS-FADD), respectively. Deletion mutants of APIP were generated by PCR and subcloned into the Xho' site of pcDNA3-HA. To generate GST-APIP protein, the CARD of Apaf-1 and {Delta}A (amino acids 16-204) of APIP were subcloned into the BamH' site of pGEX5X-1 (pGEX-CARD and pGEX-APIP).

Antibody Generation—BL21 (DE3) was transformed with pGEXAPIP and incubated with 0.3 mM isopropyl-1-thio-{alpha}-D-galactopyranoside. GST-APIP protein was purified by glutathione-SepharoseTM 4B beads (Amersham Biosciences) and injected four times into rabbit and mouse. Rabbit anti-APIP antibody was purified by antigen-affinity chromatography using SulfoLink kit (Pierce).

Northern Blot Analysis—Northern blotting was performed using multiple tissues. The Northern blot membrane was from Clontech, and a 32P-labeled APIP probe was generated by the random prime kit (Amersham Biosciences).

Reagents—Apaf-1 (-/-) and caspase-9 (-/-) MEFs were previously described (19, 20). Etoposide, cis-PLATINUM(II)-Diammine dichloride, and staurosporin were purchased from Sigma. Soluble Fas ligands were obtained from the cultured supernatant of CHO-K1-Fas cells grown in serum-free medium (CHO-S-SFM II; Invitrogen). Active specific anti-caspase-9, anti-{alpha}-tubulin, anti-rabbit IgG-horseradish peroxidase (HRP), anti-mouse IgG-HRP, anti-rat IgG-HRP, and anti-goat IgG-HRP antibodies were purchased from Santa Cruz; anti-Apaf-1 monoclonal antibody (2E12) was from Alexis (Lausen, Switzerland) (39); active specific anti-caspase-3 antibody was from Cell Signaling (Beverly, MA); anti-HA antibody was from Roche Applied Science (BM, Germany); anti-cytochrome c antibody was from BD Biosciences (Franklin Lakes, NJ); and anti-FLAG antibody was from Sigma.

In Vitro Binding Assay—The purified GST and GST-CARD coupled to glutathione-Sepharose 4B were incubated with [35S]methionine-labeled APIP, its deletion mutants, and caspase-9S by a TNT-coupled transcription/translation system from Promega for 3 h at 4 °C. After the GST pull-down assay, the precipitated proteins were washed with the binding buffer (50 mM Tris-HCl, pH 6.8, 100 mM NaCl, 1 mM dithiothreitol, 2 mM EDTA, 0.05% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 5% (v/v) glycerol), separated by 12% SDS-PAGE, and detected by autoradiogram.

Immunoprecipitation—HeLa and HEK293 cells were lysed in radio-immune precipitation assay buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml each of aprotinin, leupeptin, and pepstatin, 1 mM Na3VO4, and 1 mM NaF]. APIP was immunoprecipitated from cell lysates after incubation with anti-APIP antibody and protein G-Sepharose (Amersham Biosciences) for 3 h at 4 °C. Immunocomplexes were centrifuged, washed with cold lysis buffer at least three times, and then detected with Western blot analysis.

Stable Transfection—C2C12 cells were transfected with 1 µg of pHAAPIP using LipofectAMINE reagent from Invitrogen and then grown in selection medium containing 1 mg/ml of G418 for 2 weeks. After single cell cloning, the clones were screened by Western blot analysis.

Cell-free System—Preparation of HeLa S-100 fraction was previously described (4). Briefly, a 3-µl aliquot of the in vitro translated caspase-3 was incubated with 80 µg of S-100 fraction in the presence or absence of 1 mM dATP, 2 µg/ml cytochrome c, and 2 µg or 10 µg of purified GST or GST-APIP proteins for 1 h at 30 °C. The reaction products were subjected to 15% SDS-PAGE and exposed to x-ray film.

Fractionation of Cell Extracts—Cells were incubated in hypotonic buffer A (250 mM sucrose, 20 mM Hepes pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml each of aprotinin, leupeptin, and pepstatin, 1 mM Na3VO4, and 1 mM NaF) and lysed by homogenizer. Intact cells and nuclei were separated by centrifugation at 500 x g for 5 min; the supernatants were then centrifuged at 10,000 x g for 30 min to fractionate membrane pellet and cytoplasm.

Ischemia Induction in the Muscle of Mouse Hindlimb and TUNEL Assay—Ischemia was induced in 20-25-g male C57BL/6 mice by ligation of the femoral artery of the hindlimb. The operative procedures to generate ischemic muscle were previously described (40). The animals were anesthetized with pentobarbital intraperitoneally for the surgical procedure. The middle portion of the left hindlimb of the mouse was incised and ligated at both the proximal end of the femoral artery and the distal portion of the saphenous artery. All side and attached side branches were dissected free and then excised. The right hindlimbs of the mice were used as non-ischemic controls (n > 3, the number of mice). Mouse muscle tissues were injected with 25 µg of pAS-APIP using a glass capillary. After 2 days, unilateral hindlimb ischemia was induced for an additional day, and the affected muscles were examined for apoptosis by TUNEL analysis with ApopTag in situ apoptosis detection kits (Intergen Company, Purchase, NY).

Hypoxia and Differentiation Induction of C2C12 Skeletal Muscle Cells—C2C12 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 20% fetal bovine serum (EQUITE-Bio, Kerrville, TX) in the presence of 5% CO2. Cell differentiation of myoblasts into myotubes were induced by incubating 80% confluent C2C12 cells with 2% horse serum for 4 days. Hypoxia was achieved by culturing the cells in minimum essential medium (Invitrogen) in an air-tight Plexiglas chamber with GasPak PlusTM (BD Biosciences) as previously described (41).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation of APIP—In an attempt to identify the Apaf-1-interacting protein, we screened the Jurkat cDNA library by using the CARD of Apaf-1 as bait for a yeast two-hybrid assay. From 10 million yeast transformants, we have isolated 12 putative positive clones including Hsp70. Among them, we designated an Apaf-1-interacting protein as APIP that is also described as CGI29 and MMRP19. APIP is predicted to encode a 204 amino acid protein containing the aldolase II domain and localizes on chromosome 11p13.11-11p13.12. In GenBankTM expressed-sequenced tag data base for human, we also found an isoform of APIP, APIP2 encoding a 242 amino acid protein, which is an alternative splicing variant from a single gene and different in its N terminus from APIP (Fig. 1A). Sequence alignment shows that the amino acid sequence of APIP is highly conserved among different species. Analysis with the BLAST data base revealed that there is a single APIP gene in mice (mAPIP) that is more similar to APIP2. When examined with Northern blotting, the tissue distribution of APIP revealed a transcript of APIP with an approximate size of 1.2 kb that was ubiquitously expressed in most adult tissues with high expression in skeletal muscle, heart, and kidney (Fig. 1B).



View larger version (65K):
[in this window]
[in a new window]
  		FIG. 1.
Alignments of amino acid residues and tissue distribution of APIP. A, amino acid sequence of human APIP (hAPIP) was aligned to the sequence of mouse APIP (mAPIP), Drosophila melanogaster, and C. elegans. Identical amino acids are aligned, and conserved sequences are darkly shaded. B, APIP expression pattern was analyzed by Northern blotting using mRNA prepared from human adult tissues. The same membrane was probed with {beta}-actin cDNA as an internal control.

 
In a yeast two-hybrid assay, APIP interacted with the CARD of Apaf-1, but not with the death domain of FADD (data not shown). In vitro GST pull-down assays showed that APIP and APIP2 interacted with the GST-CARD fusion protein as efficiently as the caspase-9 short form did, but they did not interact with GST (Fig. 2A). APIP did not bind to other CARD-containing proteins such as pro-caspase-9 (data not shown). The radiolabeled APIP pulled down by GST-CARD represented about 10% of input. The endogenous interaction of APIP and Apaf-1 was examined in HeLa cells. An immunoprecipitation assay using anti-APIP antibody showed that Apaf-1 and APIP were detected in the immunoprecipitates, indicating that endogenous APIP interacts with Apaf-1 in HeLa cells (Fig. 2B, upper panel). We also were able to demonstrate an interaction of exogenous Apaf-1 with APIP by an immunoprecipitation assay of HEK293F cells transfected with HA-tagged APIP and Apaf-1 (Fig. 2B, middle panel). Intracellular interaction of APIP with the CARD of Apaf-1 was further examined after transfection with pmCARD-FLAG and pHA-APIP (Fig. 2B, lower panel). Western blot analysis with anti-FLAG antibody following immunoprecipitation with anti-HA antibody showed that APIP interacted with Apaf-1 via CARD in the cells. Sub-cellular fractionation analysis showed that APIP was found in the cytosol as Apaf-1 (data not shown).



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 2.
Interaction of APIP with the CARD region of Apaf-1. A, in vitro binding assay. GST and GST-CARD proteins were expressed and purified. Proteins bound to their affinity resins (each equivalent to 20 µg of proteins) were incubated with in vitro translated APIP protein in the presence of [35S]methionine as described under "Experimental Procedures." Caspase-9S protein was used as a positive control. After separation by 12% SDS-PAGE, the bound proteins were detected by autoradiography (upper panel), and resin-coupled proteins were visualized by Coomassie Blue staining (lower panel). Right panel represents 10% of input, radiolabeled APIP. B, intracellular interaction. Endogenous APIP protein was immunoprecipitated (IP) from HeLa cell extracts with preimmune serum (Pre) or anti-APIP antibody (APIP) (upper panel). HEK293F cells were overexpressed with Apaf-1 and HA-tagged APIP, and immunoprecipitated with anti-APIP antibody (APIP) (middle panel). Cell extracts prepared from HEK293 cells co-transfected with pmCARD-FLAG and pHA-APIP were immunoprecipitated with anti-HA antibody (HA) (lower panel). The immunoprecipitates and whole cell lysates (Lysate) were separated by SDS-PAGE and immunoblotted (IB) with the indicated antibodies. Asterisk (*) indicates heavy chain of immunoglobulin.

 
Anti-apoptotic Activity of APIP in the Mitochondria-mediated Apoptosis—Apaf-1 functions as a cytochrome c-dependent activator of caspase-9. To examine the role of APIP in cell death, we generated a C2C12 skeletal muscle cell line that stably expressed APIP (C2C12/APIP) (Fig. 3A). C2C12 or C2C12/APIP cells were exposed to various cell death stimuli. Determination of cell viability showed that overexpression of APIP suppressed apoptosis induced by etoposide, cisplatin, or staurosporin, stimuli that are known to activate the mitochondrial pathway, but APIP failed to suppress apoptosis triggered by soluble Fas ligand or TNF-{alpha} (Fig. 3A). Such inhibitory activity of APIP was also observed over 3 days in C2C12 cells and in other cells such as HeLa and HEK293 cells treated with the mitochondrial stress signals (data not shown).



View larger version (32K):
[in this window]
[in a new window]
  		FIG. 3.
Suppression of etoposide- and tBid-triggered cell death by APIP without affecting cytochrome c release from mitochondria. C2C12 or C2C12 cells stably expressing APIP (C2C12/APIP) were left untreated (CTRL) or exposed to etoposide (Etop., 25 µg/ml), cisplatin (Cisp., 100 ng/ml), staurosporin (Stau., 50 nM), TNF-{alpha} (TNF/CHX, 30 ng/ml), or soluble Fas ligand (s-Fas, 0.2 µg/ml) for 24 h. A, cell viability (% of cell death) was determined by trypan blue and TUNEL assays. B, cells exposed to etoposide (25 µg/ml) were stained with anti-cytochrome c antibody and Hoechst 33258. The arrows indicate the typical diffused and cytosolic pattern of cytochrome c and morphology of nuclei in the same cells. C, C2C12 and C2C12/APIP cells were treated with etoposide (Etop., 50 µg/ml) for 12 h. Cell extracts were separated into membrane and cytosolic fractions, and the fractionated extracts were subsequently immunoblotted with anti-cytochrome c, anti-COX4, or anti-tubulin antibody (left panel). The cell extracts were also probed with anti-active caspase-9, anti-active caspase-3, or anti-tubulin antibody (right panel). Asterisk (*) indicates nonspecific signal. D, HeLa cells were transfected with ptBid alone or together with pGFP-APIP or pGFP-APIP2 for 24 h. Cell viability was then determined based on the morphology of GFP-positive cells. Bars depict means ± S.D. from at least four independent experiments. E, wild type, Apaf-1 (-/-), or caspase-9 (-/-) MEFs were transfected with pGFP or pGFP-APIP for 24 h and then exposed to etoposide (Etop., 25 µg/ml) or TNF-{alpha} (TNF/CHX, 10 ng/ml) for 12 h. Cell viability (% of cell death) was determined by trypan blue assays of GFP-positive cells. Bars depict means ± S.D.

 
We further assessed the function of APIP in Apaf-1-mediated cell death. Immunocytochemical analysis showed that cytochrome c in the mitochondria was released into the cytosol of C2C12 cells exposed to etoposide and undergoing apoptosis (Fig. 3B). However, expression of APIP-protected C2C12 cells from etoposide-induced apoptosis despite cytochrome c release from mitochondria. Fractionation analysis followed by Western blotting also showed that cytochrome c was released from the mitochondria in both C2C12/APIP and C2C12 cells exposed to etoposide (Fig. 3C, left panel). In contrast to C2C12 cells, however, activation of caspase-9 or caspase-3, the downstream caspases of cytochrome c, was suppressed in the etoposide-treated C2C12/APIP cells (Fig. 3C, right panel). These results indicate that APIP protects cells from apoptosis by inhibiting the downstream activity of cytochrome c released from mitochondria. To explore the role of APIP as an apoptosis suppressor, HeLa cells were transiently transfected with tBid, the cleavage product of Bid by caspase-8. The tBid-triggered apoptosis was efficiently suppressed by co-expression of APIP (death rates from 62 to 35%) (Fig. 3D), indicating that APIP functions as an anti-apoptotic molecule in the mitochondria-mediated cell death pathway.

Anti-apoptotic activity of APIP was examined in Apaf-1 (-/-) or caspase-9 (-/-) mouse embryo fibroblasts (MEFs). As reported earlier, Apaf-1 (-/-) or caspase-9 (-/-) MEFs were resistant to cell death triggered by etoposide but not by TNF/CHX (Fig. 3E). Overexpression of APIP in the wild-type MEFs suppressed etoposide-induced cell death to the level of Apaf-1 (-/-) or caspase-9 (-/-) MEFs. The anti-apoptotic function of APIP was further examined by using antisense approaches. Direct targeting of APIP expression with antisense cDNA potently increased apoptosis induced by cisplatin but not by soluble Fas ligand, while antisense cDNA of FADD inhibited apoptosis in the opposite manner in HEK293 cells (Fig. 4A). Expression of antisense cDNAs of APIP in HEK293 and HeLa cells apparently diminished the expression level of APIP as determined by Western blot (Fig. 4B) and immunocytochemical analysis (data not shown), respectively. Similarly, antisense cDNA of FADD reduced expression level of FADD and was used as a control for the non-mitochondrial apoptotic signal. These results are more illustrative of the role of APIP as an anti-apoptotic molecule.



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 4.
Potentiation of cell death by antisense (AS) cDNA of APIP. A, HEK293 cells transfected with pEGFP and either pcDNA (CTRL), pAS-APIP, or pAS-FADD were exposed to cisplatin or soluble Fas ligand for 48 h. Cell viability was determined based on the morphology of GFP-positive cells. Bars depict means ± S.D. B, HEK293 cells were transiently transfected with pEGFP and either pcDNA (CTRL), pAS-APIP, or pAS-FADD for 48 h, and the expression levels of endogenous APIP and FADD were examined by Western blotting.

 
Suppression of Cytochrome c-mediated Activation of Caspase-3 and Caspase-9 —The CARD of caspase-9 binds to the CARD of Apaf-1 for its activation. As molecules interacting with the CARD of Apaf-1, competitive binding between caspase-9 and APIP to Apaf-1 was examined in vitro (Fig. 5A). In vitro GST pull-down assays showed that increasing the amounts of caspase-9 in the reaction mixtures proportionally reduced the amounts of APIP interacting with the CARD of Apaf-1, while binding of caspase-9 to the CARD of Apaf-1 gradually increased. These results indicate that APIP and caspase-9 may compete with each other for binding to Apaf-1. To examine the anti-apoptotic activity of APIP in Apaf-1-mediated activation of caspase, a cell-free system containing S-100 fraction was employed. Incubation of S-100 fraction with dATP and cytochrome c induced proteolytic activation of caspase-3 and caspase-9 (Fig. 5B). However, activation of caspase-3 and caspase-9 were suppressed by incubating the S-100 fraction with purified GST-APIP, but not with GST, suggesting that APIP inhibited the cytochrome c-mediated activation of caspase and further supporting the intracellular anti-apoptotic activity of APIP functioning downstream of the cytochrome c released from mitochondria, as shown in Fig. 3, B and C.



View larger version (33K):
[in this window]
[in a new window]
  		FIG. 5.
Suppression of cytochrome c-induced activation of caspase-3 by APIP in vitro: competitive interaction of APIP and caspase-9 with the CARD of Apaf-1. A, purified GST-CARD was incubated with 35S-labeled, in vitro translated APIP in the presence of increasing amounts of 35S-labeled, in vitro translated caspase-9. The reaction volumes and the amounts of total protein were adjusted same by adding reticulocyte lysate containing in vitro translated luciferase to the reaction mixture. After GST pull-down assay, the precipitated proteins were separated by SDS-PAGE and exposed to x-ray film. Lower graph indicates densitometric analysis of APIP protein remaining on GST-CARD. B, HeLa S-100 fraction (80 µg) was incubated with different amounts of purified GST or GST-APIP and in vitro translated, 35S-labeled caspase-3 (upper panel) or caspase-9 (lower panel) in the presence or absence of cytochrome c (2 µg/ml) and dATP (1 mM). The reaction products were separated by SDS-PAGE and exposed to x-ray film. C, schematic diagram of the APIP deletion mutants ({Delta}). Numbers indicate amino acid residues of APIP. D, in vitro binding assays were performed using purified GST-CARD and APIP deletion mutants labeled with [35S]methionine. The reaction products were fractionated by 15% SDS-PAGE and exposed to x-ray film. E, HeLa cells were transiently co-transfected with pEGFP and various deletion mutants of APIP for 24 h, and then exposed to etoposide (Etop., 25 µg/ml), cisplatin (Cisp., 100 ng/ml), or soluble Fas ligand (s-Fas, 200 ng/ml) for additional 24 h and cell viability was determined. F, purified GST-CARD was incubated with in vitro translated caspase-9 in the presence of different amounts of in vitro translated APIP or its deletion mutant proteins. The reaction volumes were adjusted same by adding reticulocyte lysate. After GST pull-down assay, the precipitated proteins were separated by SDS-PAGE and exposed to x-ray film.

 
Anti-apoptotic Activity of APIP Core Region—To map the functional domain of APIP in detail, we constructed several deletion mutants (Fig. 5C). The 35S-labeled deletion mutants of APIP were evaluated for their abilities to bind to the CARD of Apaf-1 protein in vitro (Fig. 5D). Although the {Delta}C mutant of APIP showed relatively weak interaction with Apaf-1, all deletion mutants we generated bound to GST-CARD. Subsequently, the ability of each mutant to suppress cell death was examined in HeLa cells with transient expression analysis (Fig. 5E). Interestingly, only the {Delta}A mutant containing the common core region (amino acids 16-204 of APIP) of both APIP and APIP2 was as effective as APIP in suppressing cisplatin- or etoposide-induced apoptosis, whereas other mutants showing further deletion lost their abilities to exhibit the anti-apoptotic activity. Further, in vitro competition assay between caspase-9 and either APIP or APIP deletion mutants showed that the binding of caspase-9 to the CARD of Apaf-1 was effectively decreased by the increased amounts of APIP and {Delta}A mutant, but not by {Delta}D mutant (Fig. 5F). These results indicate that the wild type and {Delta}A mutant, but not the {Delta}D mutant, of APIP compete with caspase-9 to interact with the CARD of Apaf-1, consistent with the inhibitory activities of the mutants used in Fig. 5E. These results suggest that although the N- or C-terminal region of APIP is able to interact with the CARD of Apaf-1 in vitro, a core region spanning residues 16-204 of APIP seems to be required for anti-apoptotic activity of APIP.

Block of Muscle Ischemic Damage by Induced Accumulation of APIP—Messenger RNA of APIP was abundant in skeletal muscle (Fig. 1B). To examine whether APIP was implicated in ischemic injury, the expressional regulation of APIP was investigated in the ischemia-induced mouse muscle. Western blotting and immunostaining analyses showed that interruption of the artery in the hindlimb muscle transiently induced dramatic accumulation of mAPIP, but not of Bcl-xL, cIAP1, or Bid (Fig. 6A and data not shown). The specificity of anti-APIP antibody was examined by Western blotting of muscle lysates (Fig. 6B, right panel). In contrast to the non-treated hindlimbs, the level of mAPIP protein was the highest (8-fold) at day 1 of the prolonged ischemia and returned to the control level at day 7. Interestingly, apoptotic cells were rarely observed in the ischemic muscle as examined with TUNEL assay and Hoechst dye staining (Fig. 6D). We then targeted the expression of mAPIP by directly injecting expression plasmid encoding antisense cDNA of APIP into mouse skeletal muscle. When the vector expressing green fluorescence protein was injected, green fluorescence was detected as early as day 1 and remained for at least 5 days in muscle tissues (data not shown). Ischemia-induced accumulation of mAPIP was abolished by injecting antisense cDNA of APIP into the muscle tissues (Fig. 6C). TUNEL staining detected massive dying cells in the tissue samples prepared from the hindlimb muscles injected with antisense cDNA of APIP, but not in those injected with pcDNA3 (Fig. 6D, arrowheads). These results suggest that the accumulation of mAPIP blocks ischemia-induced cell death of affected skeletal muscle.



View larger version (42K):
[in this window]
[in a new window]
  		FIG. 6.
Protection of muscle cells from ischemic damage by induced expression of APIP. Mouse muscle ischemia was induced by artery interruption of the left hindlimb and the non-ischemic, right hindlimb was used as control for the indicated times. At least three mice were investigated at each time point. The expression of mAPIP from groups of three mice was detected by Western blotting (A). The ischemic regulation of APIP expression was quantified by densitometric analysis: Data from two points were taken and averaged in the graph. (A, middle graph) or was examined by immunohistochemical analysis of tissue sections (B). The specificity of anti-APIP antibody was examined by Western blotting of two mouse hindlimb muscle lysates (B, right panel). Mouse hindlimb muscle was directly injected with pcDNA3 (CTRL) or pAS-APIP. After 2 days, the artery of the hindlimb was interrupted for additional 24 h to induce ischemic condition and the affected muscles were prepared from each for Western blot analysis (C) and TUNEL assays (D). TUNEL-positive cells indicated by arrowheads (D, upper panel) were quantified (D, lower panel).

 
To ascertain the inhibitory effects of APIP on ischemic/hypoxic damage, we examined ischemic damage of the C2C12 skeletal muscle cell line. Unlike muscle tissues, there was no change in the expression level of mAPIP in C2C12 myoblast cells under in vitro ischemic/hypoxic conditions (data not shown). While incubation of C2C12 myoblasts under hypoxic conditions for 1 day induced 50% of cell death, expression of APIP completely suppressed ischemia/hypoxia-induced death of C2C12 myoblast cells to the control level (Fig. 7A). Examination of cell extracts prepared from C2C12 and C2C12/APIP cells with Western blotting showed that hypoxia-induced activation of caspase-9 was suppressed in C2C12/APIP cells (Fig. 7B), indicating that APIP inhibits hypoxia-induced activation of caspase-9.



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 7.
Suppression of ischemic/hypoxic damage by the increased expression of APIP in C2C12 skeletal muscle cells. C2C12 cells stably transfected with pcDNA (C2C12) or pHA-APIP (C2C12/APIP) were left untreated or exposed to hypoxia for the indicated times. Cells were stained with trypan blue for the determination of cell viability (A, left graph) and for visualization at 24 h (A, right panel). C2C12 or C2C12/APIP cells were exposed to hypoxic condition for 24 h, and cell extracts were immunoblotted with anti-active caspase-9 or anti-tubulin antibody (B). C2C12 myoblast cells were incubated with 2% horse serum to induce differentiation to myotubes for 4 days. C2C12 myoblasts and myotubes were then exposed to hypoxic condition for 12 h. The cell viability of the myotube was determined by examining the morphology of multi-nuclei (n > 2) after staining with Hoechst dye (C). C2C12 myotubes were exposed to hypoxic condition for the indicated times and expression levels of mAPIP were determined by Western blotting (D). Asterisk (*) indicates nonspecific signal.

 
We then examined cell viability of C2C12 myotube cells after induction of in vitro differentiation. Interestingly, C2C12 skeletal myotubes were more resistant to hypoxic damage compared with the myoblast cells (Fig. 7C). In contrast to myoblasts, mAPIP protein was transiently induced in C2C12 myotubes in response to hypoxia (Fig. 7D), consistent with that of in vivo muscle tissues under ischemic condition and further supporting the finding that the increased expression of mAPIP protects C2C12 muscle cells from ischemia/hypoxia-induced cell death.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have identified a novel anti-apoptotic molecule in Apaf-1-mediated cell death, APIP. Apaf-1, a key regulator of apoptosis, has a major role in programmed cell death (4). In the presence of cytochrome c and dATP, Apaf-1 oligomerizes to form a very large protein complex, apoptosome (17). Apoptosome recruits and activates caspase-9, and is regulated by several factors including XIAP and heat shock proteins (25, 26, 29). Hsp70 has been reported to associate with Apaf-1 and inhibit cell death by interfering with the ability of cytochrome c and Apaf-1 to recruit caspase-9. In this study, we have provided several lines of evidence demonstrating that APIP is likely to interact with Apaf-1 and consequently inhibit Apaf-1-mediated activation of caspase-9: (i) APIP bound to Apaf-1 in vitro and in vivo; (ii) APIP inhibited cytochrome c-induced activation of caspase-9 and cell death; (iii) APIP expression suppressed apoptosis induced by the mitochondrial insults including expression of tBid, etoposide, cisplatin, or hypoxia, but not by soluble Fas ligand or TNF-{alpha}; and (iv) down-regulation of APIP expression potentiated mitochondria-mediated cell death. Suppression of etoposide-induced cell death by APIP expression was as similar as that of Apaf-1 (-/-) or caspase-9 (-/-) MEFs.

APIP negatively regulates Apaf-1-mediated cell death through a central core domain spanning the common region of APIP and APIP2. APIP and caspase-9 competed to bind to the CARD of Apaf-1 in vitro, indicating that the binding regions of caspase-9 and APIP in Apaf-1 overlap. However, additional region of Apaf-1 may participate in the protein-protein interaction with APIP because, unlike caspae-9, APIP could form complex with Apaf-1 in the absence of dATP and cytochrome c as examined with immunoprecipitation assay. There is no apparent CARD domain in APIP, which is frequently found in caspases, Apaf-1, and other CARD-containing molecules. Instead, a broad core region spanning residues 16-204 of APIP seems to be required for the inhibitory activity of APIP in Apaf-1-mediated cell death, probably through an unidentified structural motif. In that case, amino acid sequences of APIP are highly conserved from C. elegans to humans with the exception at the N terminus, showing 52% amino acid sequence homology, although two extra short stretches of APIP are found in C. elegans.

APIP may function under pathological conditions. Hypoxia is an important factor in the pathogenesis of several major diseases such as stroke and cancer (30-32). Several lines of evidence indicate that, under certain conditions, hypoxia induces cell death through the Apaf-1-mediated mitochondrial pathway (33). Hypoxia results in translocation of Bax from the cytosol to the mitochondria and activation of caspases (34-38). In addition, the cells overexpressing the anti-apoptotic Bcl-2 family proteins have been shown to prevent hypoxia-induced apoptosis by inhibiting the release of cytochrome c (34). Thus, apoptotic signals during hypoxia seem to occur through the release of cytochrome c and the Apaf-1-mediated activation of caspase-9 (36).

Interestingly, the expression level of mAPIP was highly and transiently up-regulated in the muscle tissues not showing any significant damage during ischemia. Though the difference in the expression level of mAPIP in the ischemic hindlimb muscles at day 3 may reflect the individual differences, we consistently observed transient up-regulation of mAPIP at earlier times. The observation that expression levels of mAPIP affected ischemia/hypoxia-induced death of muscle cells in vivo and in vitro led us to propose that the induction of APIP under ischemic conditions suppressed ischemic injury in the affected muscles. Thus, the observed recovery from muscle ischemic injury in mice was probably due to up-regulation of mAPIP in the affected muscle for a short period and the formation of a bypass blood vessel network in the prolonged period. The ability of APIP to prevent cell death during hypoxia is believed to be associated with Apaf-1, although it is not clear that this inhibitory effect also blocks apoptosome formation. At present, while the molecular architecture of the APIP/Apaf-1 complexes in living and dying cells remains to be determined, the binding of APIP to CARD of Apaf-1 may prevent caspase-9 from being recruited into the apoptosome under ischemia/hypoxia. Taken together, the data presented here suggest that the anti-apoptotic function of APIP lies in the inhibition of downstream activity of cytochrome c released from mitochondria during pathogenic processes including muscle ischemia, suggesting a potential means by which APIP in pathogenic cell death may be manipulated.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) BC010133 [GenBank] and NP_057041 [GenBank] .

* This work was supported by a grant from the National Research Laboratory program (to Y-K. J.) and 21 C Frontier on Brain and Functional Analysis of Human Genomics of the Korean Ministry of Science and Technology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Supported in part by the Brain Korea 21 project. Back

|| To whom correspondence should be addressed: Dept. of Life Science, Gwangju Institute of Science and Technology, 1-Oryong-dong, Buk-gu, Gwangju 500-712, Korea. Tel.: 82-62-970-2492; Fax: 82-62-970-2484; E-mail: ykjung{at}kjist.ac.kr.

1 The abbreviations used are: APIP, Apaf-1-interacting protein; AS, anti-sense; CARD, caspase recruitment domain; CHX, cycloheximide; GST, glutathione S-transferase; HA, hemagglutinin; MEFs, mouse embryo fibroblasts; TNF, tumor necrosis factor; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling; HRP, horseradish peroxidase; GFP, green fluorescent protein; GST, glutathione S-transferase; HEK, human embryonic kidney cells; FADD, fas-associated protein with death domain. Back


   ACKNOWLEDGMENTS
 
We thank Dr. H. Li (Northwestern Medical School, Chicago, IL) for ptBid, A. Memon, and I. Kim (GIST, Korea) for their critical reading of this manuscript, and Dr. D. Kim (Samsung Hospital, Korea) for technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Yuan, J., and Horvitz, H. R. (1990) Dev. Biol. 138, 33-41[CrossRef][Medline] [Order article via Infotrieve]
  2. Hengartner, M. O., Ellis, R. E., and Horvitz, H. R. (1992) Nature 356, 494-499[CrossRef][Medline] [Order article via Infotrieve]
  3. Vaux, D. L. (1997) Cell 90, 389-390[CrossRef][Medline] [Order article via Infotrieve]
  4. Zou, H., Henzel, W. J. Liu, X., Lutschg, A., and Wang, X. (1997) Cell 90, 405-413[CrossRef][Medline] [Order article via Infotrieve]
  5. Desagher, S., and Martinou, J. C. (2000) Trends Cell Biol. 10, 369-377[CrossRef][Medline] [Order article via Infotrieve]
  6. Du, C., Fang, M., Li, Y., Li, L., and Wang, X. (2000) Cell 102, 33-42[CrossRef][Medline] [Order article via Infotrieve]
  7. Kang, P. M., Haunstetter, A., Aoki, H., Usheva, A., and Izumo, S. (2000) Circ. Res. 87, 118-125[Abstract/Free Full Text]
  8. Verhagen, A. M., Ekert, P. G., Pakusch, M., Silke, J., Connolly, L. M., Reid, G. E., Moritz, R. L., Simpson, R. J., and Vaux, D. L. (2000) Cell 102, 43-53[CrossRef][Medline] [Order article via Infotrieve]
  9. Suzuki, Y., Imai, Y., Nakayama, H., Takahashi, K., Takio, K., and Takahashi, R. (2001) Mol. Cell 8, 613-621[CrossRef][Medline] [Order article via Infotrieve]
  10. van Loo, G., Saelens, X., van, Gurp, M., MacFarlane, M., Martin, S. J., and Vandenabeele, P. (2002) Cell Death Differ. 9, 1031-1042[CrossRef][Medline] [Order article via Infotrieve]
  11. Tsujimoto, Y. (2003) J. Cell. Physiol. 195, 158-167[CrossRef][Medline] [Order article via Infotrieve]
  12. Li, H., Zhu, H., Xu, C. J., and Yuan, J. (1998) Cell 94, 491-501[CrossRef][Medline] [Order article via Infotrieve]
  13. Luo, X., Budihardjo, I., Zou, H. Slaughter, C., and Wang, X. (1998) Cell 94, 481-490[CrossRef][Medline] [Order article via Infotrieve]
  14. Eskes, R., Desagher, S., Antonsson, B., and Martinou, J. C. (2000) Mol. Cell. Biol. 20, 929-935[Abstract/Free Full Text]
  15. Hu, Y., Benedict, M. A., Ding, L., and Nunez, G. (1999) EMBO J. 18, 3586-3595[CrossRef][Medline] [Order article via Infotrieve]
  16. Saleh, A., Srinivasula, S. M., Acharya, S., Fishel, R., and Alnemri, E. S. (1999) J. Biol. Chem. 274, 17941-17945[Abstract/Free Full Text]
  17. Zou, H., Li, Y., Liu, X., and Wang, X. (1999) J. Biol. Chem. 274, 11549-11556[Abstract/Free Full Text]
  18. Cecconi, F., Alvarez-Bolado, G., Meyer, B. I., Roth, K. A., and Gruss, P. (1998) Cell 94, 727-737[CrossRef][Medline] [Order article via Infotrieve]
  19. Kuida, K., Haydar, T. F., Kuan, C. Y., Gu, Y., Taya, C., Karasuyama, H., Su, M. S., Rakic, P., and Flavell, R. A. (1998) Cell 94, 325-337[CrossRef][Medline] [Order article via Infotrieve]
  20. Yoshida, H., Kong, Y. Y., Yoshida, R., Elia, A. J., Hakem, A., Hakem, R., Penninger, J. M., and Mak, T. W. (1998) Cell 94, 739-750[CrossRef][Medline] [Order article via Infotrieve]
  21. Soengas, M. S., Alarcon, R. M., Yoshida, H., Giaccia, A. J., Hakem, R., Mak, T. W., and Lowe, S. W. (1999) Science 284, 156-159[Abstract/Free Full Text]
  22. Haraguchi, M., Torii, S., Matsuzawa, S., Xie, Z., Kitada, S., Krajewski, S., Yoshida, H., Mak, T. W., and Reed J. C. (2000) J. Exp. Med. 191, 1709-1720[Abstract/Free Full Text]
  23. Marsden, V. S., O'Connor, L., O'Reilly, L. A., Silke, J., Metcalf, D., Ekert, P. G., Huang, D. C., Cecconi, F., Kuida, K., Tomaselli, K. J., Roy, S., Nicholson, D. W., Vaux, D. L., Bouillet, P., Adams, J. M., and Strasser, A. (2002) Nature 419, 634-637[CrossRef][Medline] [Order article via Infotrieve]
  24. Song, Q., Kuang, Y., Dixit, V. M., and Vincenz, C. (1999) EMBO J. 18, 167-178[CrossRef][Medline] [Order article via Infotrieve]
  25. Beere, H. M., Wolf, B. B., Cain, K., Mosser, D. D., Mahboubi, A., Kuwana, T., Tailor, P., Morimoto, R. I., Cohen, G. M., and Green, D. R. (2000) Nat. Cell Biol. 2, 469-475[CrossRef][Medline] [Order article via Infotrieve]
  26. Saleh, A., Srinivasula, S. M., Balkir, L., Robbins, P. D., and Alnemri, E. S. (2000) Nat. Cell Biol. 8, 476-483
  27. McArdle, A., and Jackson, M. J. (1997) Intracellular Mechanism Involved in Skeletal Muscle Damage, pp. 90-106, Oxford University Press, Oxford, UK
  28. Chatterjee, S., Stewart, A. S., Bish, L. T., Jayasankar, V., Kim, E. M., Pirolli, T., Burdick, J., Woo, Y. J., Gardner, T. J., and Sweeney, H. L. (2002) Circulation 106, 1212-1217
  29. Bratton, S. B., Walker, G., Srinivasula, S. M., Sun, X. M., Butterworth, M., Alnemri, E. S., and Cohen, G. M. (2001) EMBO J. 20, 998-1009[CrossRef][Medline] [Order article via Infotrieve]
  30. Saikumar, P., Dong, Z., Patel, Y., Hall, K., Hopfer, U., Weinberg, J. M., and Venkatachalam, M. A. (1998) Oncogene 17, 3401-3415[CrossRef][Medline] [Order article via Infotrieve]
  31. Marti, H. J., Bernaudin, M., Bellail, A., Schoch, H., Euler, M., Petit, E., and Risau, W. (2000) Am. J. Pathol. 156, 965-976[Abstract/Free Full Text]
  32. Semenza, G. L., Agani, F., Feldser, D., Iyer, N., Kotch, L., Laughner, E., and Yu, A. (2000) Adv. Exp. Med. Biol. 475, 123-130[Medline] [Order article via Infotrieve]
  33. Susin, S. A., Lorenzo, H. K., Zamzami, N., Marzo, I., Snow, B. E., Brothers, G. M., Mangion, J., Jacotot, E., Costantini, P., Loeffler, M., Larochette, N., Goodlett, D. R., Aebersold, R., Siderovski, D. P., Penninger, J. M., and Kroemer, G. (1999) Nature 397, 441-446[CrossRef][Medline] [Order article via Infotrieve]
  34. Shimizu, S., Eguchi, Y., Kosaka, H., Kamiike, W., Matsuda, H., and Tsujimoto, Y. (1995) Nature 374, 811-813[CrossRef][Medline] [Order article via Infotrieve]
  35. Lipton, P. (1999) Physiol. Rev. 79, 1431-1568[Abstract/Free Full Text]
  36. McClintock, D. S., Santore, M. T., Lee, V. Y., Brunelle, J., Budinger, G. R., Zong, W. X., Thompson, C. B., Hay, N., and Chandel, N. S. (2002) Mol. Cell. Biol. 22, 94-104[Abstract/Free Full Text]
  37. Santore, M. T., McClintock, D. S., Lee, V. Y., Budinger, G. R., and Chandel, N. S. (2002) Am. J. Physiol. Lung Cell Mol. Physiol. 282, 727-734
  38. Shoshani, T., Faerman, A., Mett, I., Zelin, E., Tenne, T., Gorodin, S., Moshel, Y., Elbaz, S., Budanov, A., Chajut, A., Kalinski, H., Kamer, I., Rozen, A., Mor, O., Keshet, E., Leshkowitz, D., Einat, P., Skaliter, R., and Feinstein, E. (2002) Mol. Cell. Biol. 22, 2283-2293[Abstract/Free Full Text]
  39. Hausmann, G., O'Reilly, L. A. Van, D. R., Beaumont, J. G., Strasser, A., Adams, J. M., and Huang, D. C. (2000) J. Cell Biol. 149, 623-634[Abstract/Free Full Text]
  40. Couffinhal, T., Silver, M., Zheng, L. P., Kearney, M., Witzenbichler, B., and Isner, J. M. (1998) Am. J. Pathol. 152, 1667-1679[Abstract]
  41. Malhotra, R., and Brosius, F. C, 3rd. (1999) J. Biol. Chem. 274, 12567-12575[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
R. Sanchez-Olea, S. Ortiz, O. Barreto, Q. Yang, C.-j. Xu, H. Zhu, and J. Yuan
Parcs Is a Dual Regulator of Cell Proliferation and Apaf-1 Function
J. Biol. Chem., September 5, 2008; 283(36): 24400 - 24405.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/38/39942    most recent
M405747200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cho, D.-H.
Right arrow Articles by Jung, Y.-K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cho, D.-H.
Right arrow Articles by Jung, Y.-K.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement