Humanin (HN) is a recently identified endogenous peptide that protects cells against cytotoxicity induced by various stimuli. Recently, we showed that HN binds to and inhibits Bax, a proapoptotic Bcl-2 family protein, suggesting a mechanism for HN action. In this study, we identified Bim, a Bcl-2 homology 3-only member of the Bcl-2/Bax family, as an additional HN target protein. Using in vitro protein binding, immunoprecipitation, and coimmunolocalization assays, we demonstrated that HN binds directly to the extra long isoform of Bim (BimEL) but not the long (BimL) or short (BimS) isoforms. HN also protects cells against apoptosis induced by BimEL but not BimL and BimS in gene transfection studies. In contrast, mutants of HN which failed to bind BimEL failed to protect from BimEL-induced cell death. Moreover, HN inhibited BimEL-induced release of SMAC and cytochrome c from mitochondria isolated from bax–/–cells, indicating that HN can suppress BimEL independently of its effect on Bax. Finally, we demonstrate that HN prevents BimEL-induced oligomerization of Bak using isolated mitochondria. Taken together, our results indicate that the inhibition of BimEL may contribute to the antiapoptotic properties of the HN peptide.
Apoptosis or programmed cell death is a genetically regulated cellular suicide mechanism that plays critical roles in normal development, tissue homeostasis, and elimination of infected or damaged cells. Mitochondria represent crucial organelles for the integration of various cell death stimuli and execution of the cell death program. Mitochondria are capable of releasing several apoptogenic proteins into the cytosol, including cytochrome c, AIF, SMAC/Diablo, endonuclease G, and Omi/HtrA2.
The integrity of mitochondrial membranes is controlled primarily by a balance between the antagonistic actions of the proapoptotic and antiapoptotic members of the Bcl-2 family. Bcl-2 family proteins comprise three principal subfamilies: (a) antiapoptotic members, such as Bcl-2/Bcl-X L, which possess the Bcl-2 homology (BH)1 domains, BH1, BH2, BH3, and BH4; (b) proapoptotic members, including Bax, Bak, and Bok, which have the BH1, BH2, and BH3 domains; and (c) BH3-only proteins (BOPs), such as Bid, Bim, Bad, Bik, Puma, and Bmf, which generally possess only the BH3 domain. The BH3 domain mediates interactions among Bcl-2 family proteins, allowing for networks of protein interactions. Most BOPs function as antagonists of antiapoptotic Bcl-2 family proteins. However, some BOPs operate as both antagonists of the antiapoptotic proteins and as agonists of proapoptotic family members Bax and Bak. These agonists bind via their BH3 domains, inducing oligomerization of Bax and Bak. The pore-forming capability of oligomerized Bax and Bak results in the destabilization of the mitochondrial outer membrane and the subsequent release of the death molecules from the confines of these organelles. Cells derived from bax/bak double knock-out mice are resistant to a wide spectrum of apoptotic stimuli, including BOPs, formally demonstrating that Bax and Bak mediate death signals from various BOPs.
The BOP Bim was first identified as a Bcl-2-interacting protein but was subsequently shown to bind both antiapoptotic and proapoptotic members of the Bcl-2 family. Bim is expressed in hematopoietic, epithelial, neuronal, and germ cells, and alternative mRNA splicing generates three major isoforms: short (BimS), long (BimL), and extra long (BimEL), with BimEL representing the predominant isoform in most tissues. Recent studies using gene knock-out mice have identified the bim gene as a major regulator of apoptosis in the lymphoid system. bim plays a critical role in thymic education and peripheral deletion of T lymphocytes, as well as myeloid cell homeostasis. The bim gene is also required for death of B lymphocytes in response to cross-linking of surface immunoglobulin. Bim proteins also trigger apoptosis in neurons, hematopoietic progenitors, and other types of cells, where their activity is regulated both at the level of protein expression and by post-translational modifications such as phosphorylation. In healthy cells, BimEL and BimL are sequestrated on the microtubular dynein motor complex through direct interaction with the dynein light chain LC8. In contrast, BimS does not bind to LC8, a situation that may explain the superior potency of BimS compared with BimEL and BimL in gene transfection-based apoptosis assays. Certain stress conditions, such as cytokine deprivation, γ-irradiation, and exposure to microtubule-targeting drugs, cause release of BimEL and BimL from the dynein motor complex, resulting in their translocation to mitochondria. Bim is among the few BOPs that can function as agonists of Bax and Bak, inducing oligomerization of these proteins in a BH3-dependent manner and triggering apoptogenic changes in mitochondrial membrane permeability.
By functional screening of a brain cDNA library derived from an Alzheimer disease patient, Hashimoto et al. identified an endogenous peptide, termed Humanin (HN), which suppresses neuronal cell death initiated by Alzheimer disease-related insults, including amyloid β-peptide and mutant presenilins in primary neurons and neuronal cell lines. HN is a short 24-residue polypeptide, MAPRGFSCLLLLSEIDLPVKRRA, which is apparently produced at the highest levels in testis, colon, Alzheimer disease-affected brain tissue, and some tumor cell lines. The mechanisms responsible for regulating levels of HN peptide in vivo are unknown, although post-translational control of HN protein stability by interactions with other proteins is among the reported possibilities. Interestingly, an open reading frame corresponding to HN is found embedded in a ribosome gene within the mitochondrial genome, in addition to HN-encoding genes within the nuclear genomes of several animal species, raising the possibility of gene transfer from mitochondrial to nuclear genome.
The cytoprotective mechanism of HN has been controversial. Early data suggested that HN is secreted from cells and implied that a cell surface receptor is targeted by this peptide. More recently, however, our laboratory discovered an alternative mechanism of action for HN, showing that this peptide binds to Bax and prevents the translocation of this proapoptotic protein from cytosol to mitochondria, thereby protecting cells from apoptosis-inducing insults that trigger the mitochondrial (but not nonmitochondrial) pathway for cell death. In contrast, HN does not bind multiple other Bcl-2 family members, including Bak, Bok, Bcl-2, Bcl-X L, Mcl-1, and Bcl-B. Here, we extended the protective action of HN by identifying BimEL as a new target of HN. We demonstrate that the specific binding of HN to BimEL abolishes its proapoptotic activity by preventing BimEL-induced activation of Bax and Bak and protecting cells from BimEL-induced cell death. Although the normal in vivo targets of HN remain to be clarified, these results broaden the scope of proapoptotic Bcl-2/Bax family proteins that the HN peptide is capable of antagonizing, suggesting expanded opportunities for exploiting this antiapoptotic peptide directly or indirectly for possible development of cytoprotective therapies.
HEK293T, HeLa, MEF bax+/+, MEF bax–/–, and COS-7 cells were cultured in Dulbecco's modified Eagle's high glucose medium (Irvine Scientific) containing 10% fetal bovine serum, 100 units ml–1 penicillin, and 100 μg ml–1 streptomycin at 37 °C. Transfection of cells was performed using Lipofectamine Plus reagent or Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.
pEF-PGKhygro expression plasmids incorporating the N-terminal EE (EYMPME) epitope tag and encoding BimEL, BimL, and BimS have been described previously. The plasmid pcDNA3-myc-Bcl-X L, incorporating a myc epitope tag, has also been described previously. A cDNA encoding human Bak was subcloned into the BamHI and EcoRI sites of pEGFP-C1 plasmid (Clontech). A cDNA containing the open reading frame of HN without additional flanking sequences was generated by PCR using an expressed sequence tag clone encoding full-length HN as a template (BE899497). The resulting PCR products were digested with restriction endonucleases and subcloned into the XhoI and HindIII sites of pEGFP-C1 (Clontech). Site-specific mutants of HN were created by PCR using QuikChange site-directed mutagenesis (Stratagene). For replacing cysteine 8 with proline (HN(C8P)), the following primers (XX IDT) were used: sense, 5′-CCACGAGGGTTCAGCCCTCTCTTACTTTTAACC-3′; antisense, 5′-GGTTAAAAGTAAGAGAGGGCTGAACCCTCGTGG-3′. Mutation was verified by DNA sequencing. The pRSET-BimEL plasmid encoding the recombinant His 6-tagged BimEL protein was a generous gift of Yoshide Tsujimoto.
Immunoblotting was performed as described previously, using cell lysates normalized for total protein content. For coimmunoprecipitations, cells were cultured in 50 μ m benzyloxycarbonyl-Val-Ala-Asp (O-methyl)-fluoromethylketone (ZVAD-fmk; Bachem) to prevent apoptosis. Cells were suspended in lysis buffer (50 m m Tris-HCl, pH 7.4, 150 m m NaCl, 20 m m EDTA, 50 m m NaF, 0.5% Nonidet P-40, 0.1 m m Na 3 VO 4, 20 μg ml–1 leupeptin, 20 μg ml–1 aprotinin, 1 m m dithiothreitol, and 1 m m phenylmethylsulfonyl fluoride). Lysates (500 μl) were then incubated with 1.5 μg of polyclonal Bim antibody (Sigma) or monoclonal anti-EE antibody (Babco) and 15 μl of protein G-Sepharose (Zymed Laboratories, Inc.) at 4 °C overnight. Beads were washed five times with 1 ml of lysis buffer before boiling in Laemmli sample buffer and performing SDS-PAGE, transferring to nitrocellulose membranes, and immunoblotting. Antibodies employed for antigen detection included polyclonal anti-Bim antibody (Sigma) and polyclonal and monoclonal anti-GFP antibodies (Santa Cruz).
For FPAs, various concentrations of His 6-BimEL or His 6-SMAC fusion proteins were incubated with 40 n m FITC-conjugated synthetic purified HN peptide for 10 min in the dark in phosphate-buffered saline (PBS), pH 7.4, as described previously. Fluorescence polarization was measured in PBS buffer, using an Analyst AD Assay Detection System (LJL Biosystem).
COS-7 cells were transfected with plasmids encoding pEGFP-C1, pEGFP-HN, pEFP-GK-BimEL, -BimL, or -BimS in the presence of 50 μ m ZVAD-fmk. After 48 h, cells were fixed with PBS containing 3% formaldehyde and 2% sucrose for 15 min at room temperature, permeabilized with PBS containing 0.4% Triton X-100 and 5% goat serum for 20 min at room temperature. Then, fixed cells were successively incubated with monoclonal anti-EE mAb (Babco) for 2 h at room temperature, washed with 0.1% Triton X-100 in PBS, and then incubated with Texas Red-conjugated anti-mouse antibodies (Southern Biotechnology) for 1 h at room temperature. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI).
The caspase-3-like protease activity in cell lysates was measured using a fluorometric substrate Ac-DEVD-AFC (Alexis Corporation, San Diego), which was dissolved in dimethyl sulfoxide and stored as a 10 m m solution at –20 °C. HEK293T cells were collected, washed in cold PBS, and lysed at 4 °C in buffer A (50 m m HEPES, pH 7.5, 150 m m NaCl, 20 m m EDTA, 1 m m phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 0.3% Nonidet P-40). Homogenates were centrifuged (13,000 rpm, 10 min), and the protein concentrations of the supernatants were determined using a Bradford protein assay kit (Bio-Rad). Equal amounts of supernatants (normalized for protein content) were mixed in wells of 96-well microtiter plates in a 100-μl final volume, in buffer B (50 m m HEPES, pH 7.4, 100 m m NaCl, 1 m m EDTA, 10% sucrose, 0.5% CHAPS, 5 m m dithiothreitol, and 100 μ m Ac-DEVD-AFC substrate (caspase-3/7)). Substrate cleavage was monitored continuously by spectrofluorometry, in kinetic mode, using Fluorolite 1000 (Dynatech Laboratories). Data are reported as relative fluorescence units of product produced/min/10 μg of total protein. Data were analyzed using
