Calpain-induced Bax-cleavage product is a more potent inducer of apoptotic cell death than wild-type Bax
Introduction
Apoptosis, programmed cell death, is an essential process for tissue development and homeostasis as well as for elimination of damaged cells [1]. Apoptosis is a genetically controlled process, and genes responsible for regulation of apoptosis such as Bcl-2 family members have been identified. The Bcl-2 family proteins are composed of apoptosis-promoting (Bax, Bak, Bad) and -inhibiting (Bcl-2, Bcl-xL) gene products [2]. It has been demonstrated that Bax resides in cytosol as a monomer form and migrates to mitochondria upon receiving apoptotic stimuli [3], [4], where Bax forms homo- or hetero-dimers with itself of Bcl-2/Bcl-xL. The dynamic balance between them is considered to be crucial for determining cell fate [5]. The oligomerization of Bax has been shown to favor the formation of ion-channels in liposome or planar lipid membrane [6]. Such ion channels can participate in the release of cytochrome c from mitochondria, where cytochrome c causes activation of caspase-9 through formation of pro-caspase-9/Apaf-1 complex in the presence of adenosine triphosphate, resulting in the activation of executor caspases. Bax in combination with a voltage-dependent anion channel forms a big channel [7], which could allow cytochrome c to migrate into cytosol. Moreover, Bax is also a component of permeability transition pore complexes constituting a megachannel, composed of adenine nucleotide translocator [8]. Bcl-xL or Bcl-2 is proposed to prevent apoptosis through association of or independent of Bax [5].
Wild-type (wt) p21 Bax has been shown to undergo post-translational modification during apoptosis. For example, p18 Bax generation through wt Bax cleavage has been observed in response to various stimuli such as Interferon (IFN)-α [9] and chemotherapeutic drugs [10]. The p18 Bax generation appeared to correlate with the induction and/or amplification of apoptotic cell death [10], [11], [12], [13], [14], [15]. Moreover, Wood et al. have recently demonstrated that Ca2+-dependent cysteine protease calpain is responsible for cleavage of wt Bax [10], the site of which is suggested to be located in the N-terminal region, probably around amino acids 30–33.
The mechanisms by which wt Bax initiates and/or amplifies apoptosis still remain largely unclear. In the present study, we clarified the cleavage site of wt Bax by calpain using Glutathione S-transferase (GST)-Bax fusion protein. Sequencing analysis of the COOH portion of the fusion protein revealed that the cleavage site is at the glutamine after Leu-Leu-Leu-Gln (LLLQ). The truncated form of Bax (tBax29) functioned as a more potent inducer of apoptotic cell death than the wt form of Bax. Taken together, the tBax29 would have some implications for the analysis of Bax-mediated apoptotic events, and also be useful for the treatment of malignant tumors.
Section snippets
Expression and purification of GST-Bax (ΔTM) fusion protein
Bax lacking possible transmembrane domain (ΔTM) was generated by polymerase chain reaction (PCR) amplification using human full-length Bax cDNA (a gift from Dr S. J. Korsmeyer; Howard Hughes Medical Institute (HHMI) and Washington University, St. Louis, MO) as a template, and then cloned in EcoRI/XhoI site of pGEX-4T vector containing GST in frame at the N terminus. The GST-Bax plasmids were transformed into bacteria BL21 (DE3) cells, and transformed cells were grown in LB medium in the
Calpain-induced cleavage of GST-Bax in vitro
To determine the cleavage site of Bax by calpain, GST-tagged Bax lacking possible mitochondrial targeting domain (ΔTM), COOH 169–192 amino acids, was prepared. The cDNA encoding GST was fused with the 5′ termini of cDNA encoding Bax lacking 75 nucleotides at 3′ termini and was transformed into bacteria cells, followed by induction with IPTG. The lysate from the bacteria was affinity purified with glutathione Sepharose 4B. The GST-Bax (ΔTM), the 46 kDa protein, was treated with calpain or
Discussion
Bcl-2 family proteins play a crucial role for regulation of apoptotic processes; some members including Bax and Bak function as pro-apoptotic proteins, while others including Bcl-xL and Bcl-2 function as anti-apoptotic proteins [2], [23], [24]. Pro-apoptotic protein Bax has been cleaved at the NH2-terminal region leading to p18 Bax generation during a late phase of apoptotic processes [9], [10], [25], [26], which might reflect an amplification loop [27]. Alternatively, calpain-mediated
Acknowledgements
We thank Dr Stanley J. Korsmeyer (Washington University, St Louis, MO) and T. Watanabe (Department of Pathology, Institute of Medical Science, University of Tokyo, Japan) for providing human Bax cDNA and pMEG vectors, respectively. The authors are indebted to Prof. J. Patrick Barron of the International Medical Communications Center of Tokyo Medical University for his review of this manuscript.
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