Bcl2-L-10, a novel anti-apoptotic member of the Bcl-2 family, blocks apoptosis in the mitochondria death pathway but not in the death receptor pathway

Hong Zhang 0 Wolfgang Holzgreve 0 Christian De Geyter 0 0 University Women's Hospital Basel , Schanzenstrasse 46, CH-4031 Basel, Switzerland DDBJ/EMBL/GenBank accession no. AF285092 By GenBank database searches and PCR, we have identified a novel human Bcl2-like gene, Bcl2-L-10, which contains conserved BH4, BH1 and BH2 domains but lacks BH3 domain. The Bcl2-L-10 gene has been assigned to chromosome 15q21.2. Transfection experiments demonstrated that Bcl2-L-10 can block apoptosis induced by interleukin-3 withdrawal and Bax expression, by prevention of cytochrome C release, caspase-3 activation and mitochondrial membrane potential collapse. Bcl2-L-10 cannot block TNF-induced apoptosis. Furthermore, both the BH4 domain and the transmembrane domain of Bcl2-L-10 are necessary for its suppressive action on cell death. Our results demonstrated that Bcl2-L-10 is a newly detected anti-apoptotic member of the Bcl-2 family and that it blocks apoptosis in the mitochondrial death pathway but not in the death receptor pathway. - Programmed cell death or apoptosis plays an essential role in the development of multicellular organisms and in pathological processes (for reviews see 1,2). Dysregulation of apoptosis is involved in the development of many diseases such as cancer, autoimmunity and various degenerative diseases. Proteins of the Bcl-2 gene family, which are evolutionarily conserved from the Sponges to man (3), are the most prominent regulators of apoptosis and are of considerable importance for clinical medicine. The Bcl-2 family consists of a growing number of proteins, which contain four conserved Bcl-2 homology domains (BH1, BH2, BH3 and BH4), together with a transmembrane domain, all being identified as crucial for the regulation of apoptosis. Based on functional studies and the retention of BH domains, the Bcl-2 family can be divided into three subgroups (1). The Bcl-2 subgroup includes all anti-apoptotic proteins, such as Bcl-2, Bcl-xL, A1/Bfl-1 and Mcl-1. The Bax subgroup consists of pro-apoptosis members, such as Bax, Bak and Bad. Both groups contain more than one BH domain. The third subgroup contains BH3-only proteins, such as Bid and Bim, which can interact with either anti-apoptotic proteins or proapoptosis members and promote apoptosis (4). The observation that supressors and promoters of cell death interact with each other by forming homodimers or heterodimers suggests that apoptosis is regulated, at least in part, by proteinprotein interaction (5,6). Despite the clear cut functions of the distinctive Bcl-2 family proteins either in the promotion or in the suppression of apoptosis, they play non-redundant roles in normal embryonic development, in tissue homeostasis and in a multitude of pathological processes. Both immunohistochemical and mRNA analysis have shown consistently that the expression patterns of the Bcl-2 gene family proteins are distinctive, albeit with certain overlappings, suggesting that the Bcl-2 gene family proteins regulate cell death at specific stages of cell differentiation through tissue-specific control of their expression (710). Gene knockout studies further demonstrated that the Bcl-2 gene family proteins play different roles in embryogenesis (1120). Therefore, identification of all Bcl2-like proteins will permit us to understand the structure, the function and the mutual functional relationships of the various members of the Bcl2 family, which control apoptosis in vivo. Bcl2-L-10 is a novel human Bcl-2 related gene In order to isolate novel Bcl-2 related genes, TBLASTN was used to screen public GenBank database with the amino acid sequences of all known Bcl-2 family members from various species (see Materials and Methods). Two short sequences (GenBank accession nos AA005293 and AA098865), which were identical and shared significant amino acid sequence similarities with the BH1 domain of Bcl-2 family members, were identified from the GenBank database expressed sequence tags (ESTs). Subsequently, the nucleotide sequence of the ESTs was used as a probe to query search the Human Genome Project DataBase, which revealed a matching genome sequence. Conceptually translation analysis revealed that the deduced amino acid sequence contains an open reading frame (ORF) of 204 amino acids, which we termed Bcl2-L-10 and considered as a Bcl2-like-10 protein. Accordingly, the 5 cDNA sequence was then obtained by PCR using the human placenta cDNA as a template (Materials and Methods). The full cDNA sequence contains a 5-untranslated region (5-UTR) of 80 bp and a 3-UTR of 581 bp, including a poly(A)+ signal (Fig. 1A). The deduced amino acid sequence corresponded to a 23 kDa polypeptide. The coding sequence thus depicts an N-terminal region of 13 amino acids (residues 1729) with homology to a BH4 domain, BH1 and BH2 domains and a C-terminal region (residues 183199) being a putative membrane anchor sequence present in many anti-apoptotic members (1,21) (Fig. 1C). The Bcl2-L-10 amino acid sequence also contains a potential PEST sequence, a domain rich in proline, glutamate, serine, threonine and aspartate residues (22), and an amino acid stretch (LAAS), which has not been found in any other Bcl2-like proteins (Fig. 1C). Comparison of the primary structure of Bcl2-L-10 and other Bcl-2 related proteins (23) revealed that the amino acid sequence of Bcl2-L-10 showed 49% identity with mouse Boo/ Diva (24,25) and 37% identity with avian NR-13 (26). We view Bcl2-L-10 as an evolutionarily distinctive gene but related to other members (Fig. 1B). We analyzed the predicted secondary structure of Bcl2-L-10 by NNPREDICT, a protein secondary structure prediction program (27) and compared it with Bcl-xL, Bcl-2 and Bax, of which the three dimensional structures have been determined (2830). We found that Bcl2-L-10 also has eight predicted -helices similar to Bcl-xL, Bcl-2 and Bax (Fig. 1C). The Bcl2-L-10 gene is localized at chromosomal 15q21 Comparison of the cloned cDNA sequence with the genome sequence deposited in the Human Genomic Database revealed the genomic structure and the chromosomal localization of the Bcl2-L-10 gene. As shown in Figure 1A, the gene consists of two exons. The intron/exon junctions followed the GT/AG rule and were flanked by conserved sequences (data not shown). This gene is located on human chromosome 15q21.2 within human genomic bacterial artificial chromosome (BAC) clone CTD-2184D3 (GenBank accession no. AC023906) by using the Human Genome Browser program. To compare the tissue distribution of Bcl2-L-10 mRNA expression with other Bcl-2 related genes, we have used a semiquantitive PCR-based system, the Rapid-Scan gene expression panel, to profile Bcl2-L-10 mRNA expression in 24 different human organ tissues. In contrast to the relatively restricted expression of Boo/Diva mRNA in the ovary (24,25), Bcl2-L-10 mRNA appeared to be more widely expressed. While Bcl2-L-10 expression could be detected in the brain, heart, kidney, spleen, li (...truncated)