Apolipoprotein L6, a Novel Proapoptotic Bcl-2 Homology 3–Only Protein, Induces Mitochondria-Mediated Apoptosis in Cancer Cells Zhihe Liu,1 Huimei Lu,1 Zeyu Jiang,2 Andrzej Pastuszyn,1 and Chien-an A. Hu1 1Department of Biochemistry and Molecular Biology and 2Division of Biocomputing, University of New Mexico School of Medicine, Albuquerque, New Mexico Abstract Introduction Cancer cells frequently possess defects in the genetic Apoptosis is a complex and highly regulated cell death and biochemical pathways of apoptosis. Members of the process that can be distinguished by cellular and biochemical Bcl-2 family play pivotal roles in regulating apoptosis hallmarks, including release of apoptogenic factors, activation and possess at least one of four Bcl-2 homology (BH) of caspases, chromatin condensation, and membrane blebbing. domains, designated BH1 to BH4. The BH3 domain is This cell death pathway is used by multicellular organisms to the only one conserved in proapoptotic BH3-only eliminate unwanted or injured cells and is critically important proteins and plays an important role in protein-protein for maintaining homeostasis during development and through- interactions in apoptosis by regulating homodimerization out adulthood in animals (1-4). Dysregulation of apoptosis is and heterodimerization of the Bcl-2 family members. evident in many human diseases, including cancer (5) and To date, 10 BH3-only proapoptotic proteins have been neurodegenerative disorders (6). identified and characterized in the human genome. Importantly, in mammals, there are at least three distinct but The completion of the Human Genome Project and the interactive and interconnected apoptotic pathways: mitochon- availability of various public databases and sequence dria-mediated, death receptor–initiated, and endoplasmic retic- analysis algorithms allowed us to use the bioinformatic ulum stress-mediated pathways (1, 2, 7). Activation of caspases, database-mining approach to identify one novel a group of cysteine-activated aspartate-specific proteases, is a BH3-only protein, apolipoprotein L6 (ApoL6). The common characteristic of all three apoptotic pathways (8, 9). In full-length cDNA of ApoL6 was identified, cloned, and general, activation of caspase-9 is associated with mitochondria- functionally expressed in p53-null colorectal cancer mediated apoptosis, whereas activation of caspase-8 frequently cells (DLD-1). We found that overexpression of wild-type mediates death receptor–regulated apoptosis. A connection ApoL6 induced mitochondria-mediated apoptosis in between death receptor–initiated and mitochondria-mediated DLD-1 cells characterized by release of cytochrome c pathways is made by caspase-8-cleaved and caspase-8-activated and Smac/DIABLO from mitochondria and activation of Bid, a proapoptotic member of the Bcl-2 family (1, 8, 9). The caspase-9, whereas ApoL6 BH3 domain deletion allele Bcl-2 family members play pivotal roles in regulating and did not. In addition, overexpression of ApoL6 also interconnecting all three apoptotic pathways in which they induced activation of caspase-8. Furthermore, we can either promote cell survival (antiapoptotic; e.g., Bcl-2, and showed that adenovirus harboring the full-length cDNA Bcl-xL) or induce cell death (proapoptotic; e.g., Bax, Bad, Bid, of ApoL6 induced marked apoptosis in a variety of Noxa, and PUMA). It is well recognized that cancer cells often cancer cell types, and ApoL6 recruited and interacted show altered ratios of antiapoptotic members to proapoptotic with lipid/fatty acid components during the induction members of the Bcl-2 family. Disruption of homeostasis of the of apoptosis. To our knowledge, this is the first Bcl-2 family members promotes survival and confers resistance example that intracellular overproduction of an to therapy in cancer (5, 10, 11). Each member of the Bcl-2 apolipoprotein induces marked apoptosis. (Mol Cancer protein family contains at least one to four conserved protein Res 2005;3(1):21–31) sequences, Bcl-2 homology (BH) domains designated BH1 to BH4 (10-12). Proapoptotic ‘‘BH3-only’’ proteins contain only the BH3 homology sequence attesting to the functional significance of this domain. It has been shown that the BH3- Received 9/20/04; revised 12/6/04; accepted 12/8/04. only proteins play important roles in protein-protein interac- Grant support: Howard Hughes Medical Institute research aids to University of tions, regulation of dimerization, and protein-permeable pore New Mexico Cancer Research and Treatment Center, American Cancer Society formation of the Bcl-2 family members in apoptosis (10-12). ACS-IRG-192 grant 412488-00095, and University of New Mexico Research Allocation Committee grant C-2222-RAC (C-A.A. Hu). BH3-only proteins function either as sensors or as second The costs of publication of this article were defrayed in part by the payment of messengers of discrete apoptotic stimuli and can interact with page charges. This article must therefore be hereby marked advertisement in the multidomain Bcl-2 family proteins to either antagonize or accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Chien-an A. Hu, Department of Biochemistry activate their function in apoptosis (10, 11, 13). They have been and Molecular Biology, MSC08 4670, 1 University of New Mexico, shown to induce apoptosis by binding to antiapoptotic Bcl-2 Albuquerque, NM 87131-0001. Phone: 505-272-8816; Fax: 505-272-6587. E-mail: [email protected] family members to inhibit their activity (10, 14) and other Copyright D 2005 American Association for Cancer Research. proapoptotic family members to enhance their activity (15, 16). Mol Cancer Res 2005;3(1). January 2005 21 Downloaded from mcr.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. 22 Liu et al. It is generally accepted that BH3-only proteins require the Moreover, we show that ApoL6 induces a BH3-dependent multidomain proapoptotic Bcl-2 family members to induce cell apoptosis and interacts with lipid/fatty acid components during death (7, 16, 17). Two lines of evidence indicate that BH3 the induction of apoptosis, suggesting its role in connecting domain–mediated protein-protein interactions are determinants lipid second messengers and cell death. of proapoptotic activity of BH3-only proteins. First, deletion alleles of the BH3 domain of some of the BH3-only proteins (e.g., PUMA and Bad) failed to induce apoptosis (18, 19). Results Second, peptides derived from BH3 domains of the BH3-only Employing a database search of open reading frame based proteins can induce apoptosis by binding to the hydrophobic on 9-residue consensus sequence constructed from 17 known pocket of the antiapoptotic proteins or by ‘‘sensitizing’’ cells to human Bcl-2 protein sequences (Fig. 1), we identified a apoptotic stimuli (14, 19). In addition, BH3-only proteins have BH3 domain embedded with the ApoL6 gene (Genbank been shown to interact with intrinsic mitochondrial membrane accession no. NM_030641). ApoL proteins belong to a newly proteins, such as the voltage-dependent anion channel or the identified, high-density lipoprotein family composed of six adenine nucleotide exchanger, to induce mitochondrial dys- members (L1-L6) that share significant sequence identity function and cytochrome c release (13, 20, 21). Importantly, within the predicted amphipathic a-helix. Evidence suggested several genes encoding BH3-only proteins (e.g., Bid, Noxa, and that these proteins play a central role in cholesterol transport PUMA) are p53 downstream targets in p53-induced apoptosis (24-29). Previous studies showed that genes encoding ApoL are and are expressed in overlapping as well as unique and specific localized to human chromosome 22q12-13 (24-26). A compar- tissues and cells (3, 10-12, 22). Gene knockout studies in mice ison of gene expression at the RNA level with ApoA-I, the have shown unique, specific roles for various Bcl-2 family major structural component of high-density lipoprotein, sug- genes in regulating cell proliferation and death in specific gests that the ApoL proteins play general and fundamental roles tissues/cells or under particular developmental, physiologic, or in lipid biochemistry (26, 27). Previous studies also showed that pathologic circumstances (16, 17, 23). Finally, the various ApoL6 is a widely expressed lipid binding protein (refs. 24-29; members of the Bcl-2 family are localized in different also in GeneCards database). We found that full-length ApoL6 subcellular compartments, thereby indicating their nonredun- cDNA could be successfully amplified from a variety of human dant roles in physiologic and pathologic states (10-12, 22, 23). cell lines, including DLD-1 (colorectal), HEK 293 (human Therefore, it is important to identify all members of the Bcl-2 embryonic kidney), and DU-145 (prostate) cells (data not family, particularly the BH3-only proteins, and to delineate the shown). Previous studies have predicted that extracellular cellular and biochemical contexts in which they contribute to ApoL6 is a protein component of high-density lipoprotein, the regulation of apoptosis. Currently, 10 BH3-only proapop- whereas intracellular ApoL6 may affect the movement of lipids totic proteins have been identified (7, 10-12, 14). Through or allow the binding of lipids to organelles (27, 30, 31). Human a database-mining approach, we identified and characterized ApoL6 encodes a 343–amino acid protein and has a calculated apolipoprotein L6 (ApoL6) as a novel BH3-only protein.