Biochemical and Biophysical Research Communications
Molecular interaction between HAX-1 and XIAP inhibits apoptosis
Introduction
Apoptosis is a complex biological process that kills unwanted cells during animal development, normal homeostasis and immune response. Apoptosis is mediated by a family of aspartate-specific cystein protease known as caspase [1]. Caspases play a pivotal role in both the initiation and the execution of apoptosis [2]. Among the caspase family, caspase-3, an effector caspase, plays a crucial role in the execution of apoptosis. It is activated by an initiator caspases such as caspase-9 through cleavage at a specific internal aspartate residue [2]. Activated caspase-3 cleaves downstream substrates such as molecules involved in DNA repair, apoptosis inhibitors and signaling proteins. To date, a number of proteins have been identified as substrates of caspase-3. Recently, we demonstrated that HAX-1 is cleaved by caspase-3 during apoptosis [3].
HAX-1 was first identified to associate with hematopoietic cell-specific Lyn substrate 1 (HS1), which is a component of the B-cell receptor signaling pathway [4]. Subsequently, HAX-1 was shown to interact with a diverse group of proteins including phospholamban [5], polycystic kidney disease protein PKD2 [6], SERCA2 [7], Epstein–Barr virus nuclear antigen leader protein (EBNA-LP) [8], K15 protein of Kaposi’s sarcoma-associated herpes virus [9], Omi/HtrA2 serine protease [10] and caspase-9 [11]. HAX-1 is an anti-apoptotic protein that shares homology with the BH1 and BH2 domains from the Bcl-2 family proteins [12]. Although it has been reported that HAX-1 is involved in multiple cellular functions such as cell survival and calcium homeostasis in cardiac tissue [12], the detailed functional mechanism of HAX-1 remains unclear. Therefore, the identification of new binding partners is important for understanding the functional mechanism of HAX-1.
The inhibitor of apoptosis protein (IAP) family serves as critical checkpoints of apoptotic cell death. IAP family members including X-linked inhibitor of apoptosis protein (XIAP), c-IAP1, c-IAP2 and ML-IAP, have been well characterized as inhibitors of caspases [13]. Among the IAP family members, XIAP is the most potent suppressor of apoptosis. XIAP blocks the apoptotic pathway by binding and inhibiting caspase-3, -7 and -9 [11]. XIAP contains three tandem baculovirus IAP repeat (BIR) domains followed by a RING zinc finger domain. Previous biochemical studies demonstrated that a region encompassing the second BIR domain (BIR2) is involved in the suppression of caspase-3 and caspase-7, while the third BIR domain (BIR3) binds to and inhibits caspase-9 [14]. Through its RING zinc finger domain, XIAP can function as an ubiquitin ligase both to itself and other target proteins, which are then degraded in the proteasome [15]. During apoptosis, the caspase-inhibiting function of XIAP can be antagonized by Smac/Diablo [16] and Omi/HtrA2 [10].
In this study, we identified XIAP as a novel binding partner of HAX-1 by a combination of immunoprecipitation (IP) and 2D gel electrophoresis (2-DE). By performing various biochemical experiments, we characterized the molecular interaction between HAX-1 and XIAP. First, GST pull-down assays showed that HAX-1 binds to the BIR2 and BIR3 domains of XIAP whereas XIAP binds to the C-terminal domain of HAX-1. Second, surface plasma resonance experiments demonstrated that the BIR2 and BIR3 domains of XIAP bind to HAX-1 with similar affinity. Third, ubiquitination and cell viability assays showed that the interaction between HAX-1 and XIAP contributes to cell survival by blocking the polyubiquitination of XIAP, thereby stabilizing XIAP.
Section snippets
Materials and methods
Immunoprecipitation for 2-DE. For identification of novel HAX-1 interacting proteins, pFLAG-CMV-C2 and FLAG tagged HAX-1 plasmids were transfected into Bosc 23 cells. After 48 h of transfection, the cells were harvested and lysed in Nonidet P-40 lysis buffer (150 mM NaCl, 20 mM Tris–HCl, pH 7.5, 1% Nonidet P-40 and 1% glycerol). Lysates (3.5 mg) were mixed with 15 μl of anti-FLAG M2-agarose affinity gel and incubated overnight at 4 °C. The antigen–antibody complex were collected by centrifugation at
Proteomic identification for mining novel HAX-1 binding proteins
To identify the HAX-1 interactome, we performed proteomics-based screening using a combination of IP and 2-DE. Bosc 23 cells were transfected with FLAG-tagged HAX-1 expression plasmid or FLAG control vector, and the HAX-1 protein was then isolated by IP. After IP, the eluted proteins were applied to 2-DE to separate the candidate HAX-1-interacting partners. Subsequently, the 2-DE gel was visualized by silver staining and the protein spots were analyzed by PDQuest-2-DE analysis software. Protein
Acknowledgments
This study was supported by a grant from Korea Science and Engineering Foundation (Basic Research Program No. 2008-01050) (to S.G. Park) and a grant from the National R&D Program for Cancer Control, Ministry for Health, Welfare and Family Affairs, Republic of Korea (0720130) (to S.-W. Chi). This study was also supported by a grant from KRIBB Research Initiative Program.
References (27)
- et al.
HS 1-associated protein X-1 is cleaved by caspase-3 during apoptosis
Mol. Cells
(2008) - et al.
Phospholamban interacts with HAX-1, a mitochondrial protein with anti-apoptotic function
J. Mol. Biol.
(2007) - et al.
Regulation of HAX-1 anti-apoptotic protein by Omi/HtrA2 protease during cell death
J. Biol. Chem.
(2004) - et al.
HAX-1: a multifunctional protein with emerging roles in human disease
Biochim. Biophys. Acta
(2009) - et al.
IAPs: what’s in a name?
Mol. Cell
(2008) - et al.
A single BIR domain of XIAP sufficient for inhibiting caspases
J. Biol. Chem.
(1998) - et al.
Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition
Cell
(2000) - et al.
NMR structure and mutagenesis of the third BIR domain of the inhibitor of apoptosis protein XIAP
J. Biol. Chem.
(2000) - et al.
An IAP–IAP complex inhibits apoptosis
J. Biol. Chem.
(2004) - et al.
Smac3, a novel Smac/DIABLO splicing variant, attenuates the stability and apoptosis-inhibiting activity of X-linked inhibitor of apoptosis protein
J. Biol. Chem.
(2003)
Antagonizing XIAP-mediated caspase-3 inhibition. Achilles’ heel of cancers?
Cancer Cell
Small-molecule antagonists of apoptosis suppressor XIAP exhibit broad antitumor activity
Cancer Cell
Caspases: enemies within
Science
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2014, FEBS LettersCitation Excerpt :In addition, HAX-1 also appears to resemble the inhibitor of apoptosis (IAP) proteins, a family of endogenous caspase inhibitory proteins, at least to some degree. Hence, overexpression of HAX-1 was shown to inhibit caspase-9 in cardiomyocytes [10], and HAX-1 was recently shown to bind to X-linked IAP (XIAP) and to suppress the polyubiquitination of XIAP [11], presumably leading to apoptosis inhibition by reducing the proteosomal degradation of XIAP, in analogy with other IAP-IAP complexes [12]. Furthermore, HAX-1 is cleaved in mitochondria by Omi/HTRA2 [13], a serine protease known to bind IAPs in the cytosol [14].
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These authors contributed equally to this work.