BIOLOGICAL OVERVIEW

Bcl-2 family proteins are key regulators of apoptosis. Both pro-apoptotic and anti-apoptotic members of this family are found in mammalian cells, but only the pro-apoptotic protein Debcl has been characterized in Drosophila. Buffy, the second Drosophila Bcl-2-like protein, is a pro-survival protein. Ablation of Buffy by RNA interference leads to ectopic apoptosis, whereas overexpression of buffy results in the inhibition of developmental programmed cell death and gamma irradiation-induced apoptosis. Buffy interacts genetically and physically with Debcl to suppress Debcl-induced cell death. Genetic interactions suggest that Buffy acts downstream of Reaper, Grim and Head involution defective, and upstream of the apical caspase Dronc. Furthermore, overexpression of buffy inhibits ectopic cell death in diap1 (th⁵) mutants. Taken together these data suggest that Buffy can act downstream of Rpr, Grim and Hid to block caspase-dependent cell death. Overexpression of Buffy in the embryo results in inhibition of the cell cycle, consistent with a G₁/early-S phase arrest. These data suggest that Buffy is functionally similar to the mammalian pro-survival Bcl-2 family of proteins (Quinn, 2003).

Accumulated evidence suggests that in mammalian cells, mitochondrial Bcl-2 prevents the release of cytochrome c, required for the formation of the Apaf-1 apoptosome and therefore caspase activation. Conversely, the pro-apoptotic Bcl-2 proteins promote mitochondrial permeability and cytochrome c release. Life or death of the cell is determined by whether the balance is tipped towards the pro-survival or the pro-apoptotic Bcl-2 members. In Drosophila, there are two homologs of the Bcl-2/Ced-9 family of programmed cell death (PCD) proteins, Debcl/dBorg-1/dRob-1 and Buffy/dBorg-2. Although both Debcl and Buffy share the BH1, BH2, BH3 and C-terminal transmembrane domains of the Bcl-2 family of proteins, they appear to lack the N terminal BH4 domain. In mammals, the BH4 domain distinguishes the pro-apoptotic Bcl-2 family members, e.g. Bax and Bok, from the anti-apoptotic members, e.g. Bcl-2, Bcl-xL and Bcl-w. Based upon this, both Debcl and Buffy were expected to be pro-apoptotic. Although both Debcl and Buffy are most closely related to the mammalian pro-apoptotic Bok, only Debcl has been shown to have a pro-apoptotic function in Drosophila, since ectopic overexpression of debcl in transgenic flies results in ectopic PCD and functional knockout of Debcl by RNA interference (RNAi) leads to an inhibition of cell death. Buffy is a pro-survival relative of Bcl-2/Ced-9. Buffy is required for cell survival and can prevent developmental and irradiation-induced cell death. Also, Buffy overexpression prevents cell cycle progression and results in the accumulation of cells in G₁, like its mammalian pro-survival counterpart Bcl-2. Thus, both pro-survival and cell cycle functions of Bcl-2 have been evolutionarily conserved in Buffy, suggesting that Buffy is the Drosophila homolog of the pro-survival Bcl-2 proteins (Quinn, 2003).

It is concluded that Buffy functions in a similar manner to the pro-survival mammalian Bcl-2 proteins since it (1) is required for cell survival, (2) inhibits developmentally regulated apoptosis, (3) inhibits gamma-irradiation-induced apoptosis, (4) binds the Drosophila pro-apoptotic Bcl-2 homolog Debcl, and can suppress Debcl-induced cell death, and (5) when overexpressed has an inhibitory effect on cell cycle progression (Quinn, 2003).

In mammals there are multiple pro-survival Bcl-2 proteins that play a tissue-specific role in protecting cells from apoptosis. Therefore, targeted knockout of individual pro-survival Bcl-2 members does not result in the death of the entire organism, suggesting that other pro-survival Bcl-2 proteins can compensate. For example, knockout studies show that murine Bcl-2 is required for survival of stem cells from kidney and melanocytes and adult lymphocytes, while Bcl-X_L is required for survival of neuronal and erythroid cells. In contrast, RNAi knockdown of buffy results in general embryonic cell death, suggesting that Buffy is a principle cell survival protein at this stage of Drosophila development. Indeed, database analysis shows no other Bcl-2-related proteins, apart from Buffy and Debcl (Quinn, 2003).

Overexpression of Bcl-2 impairs the apoptotic response to DNA damaging agents such as ionizing radiation. One of the reasons many tumors are resistant to chemotherapy and radiation therapy is that they mis-express Bcl-2. In the fly, increased levels of Buffy are sufficient to inhibit the Drosophila apoptotic pathway that normally responds to DNA damaging agents (Quinn, 2003).

Overexpression of mammalian Bcl-2 in Drosophila tissues has been shown to inhibit apoptosis, induced by either irradiation or Rpr overexpression (Gaumer, 2000; Brun, 2002). As has been found for Buffy, genetic analysis places the anti-apoptotic activity of Bcl-2 downstream of Rpr when expressed in flies (Brun, 2002). This is most likely a consequence of Bcl-2 protein binding to and sequestering Debcl (Colussi, 2000). In mammals, Bcl-2-mediated inhibition of apoptosis requires an alpha-helical domain within the N-terminal BH4 domain (Hunter, 1996). In contrast, Buffy’s N-terminus, and the putative alpha-helices therein, were not required for either inhibition of irradiation-induced apoptosis or suppression of Rpr-, Grim- and Hid-induced apoptosis. Thus, the C-terminal region containing the BH1, BH2, BH3 and membrane anchor is sufficient for Buffy’s cell survival function (Quinn, 2003).

Certain factors controlling cell cycle progression are also sensitive to apoptotic stimuli. Indeed, cell cycle factors may promote apoptosis under conditions unfavorable for proliferation, thus rendering cycling cells more vulnerable to apoptosis. Evidence that Bcl-2 plays a role in controlling cell cycle progression has been accumulating steadily, and in this study the first in vivo evidence is provided that a member of the Bcl-2 family can result in cell cycle inhibition. However, in contrast to the serum-deprived G0 cells that have been used previously, Drosophila embryonic cells cycle normally prior to overexpression of Buffy (Quinn, 2003 and references therein).

The cell cycle delay that occurs as a consequence of Buffy overexpression is dose dependent. Expression of two copies of UAS-buffy via en-GAL4 is embryonic lethal, presumably as a consequence of the G₁-S cell cycle arrest, which results in less cells in the En-stripes and insufficient cells to complete embryonic development. Two copies of en-GAL4,UAS-buffy (i.e., high level of Buffy protein) induce a cell cycle arrest and are embryonic lethal; however, flies expressing one copy (lower level of expression) are viable. Indeed, a low level of ubiquitous Buffy expression does not cause a cell cycle arrest, but results in the production of additional neural cells. Thus, the effect of Buffy overexpression is dose dependent; high levels of Buffy overexpression induce cell cycle arrest, which will ultimately result in fewer cells, while lower levels can inhibit developmental cell death and are associated with increased cell numbers (Quinn, 2003).

Consistent with cell cycle arrest, Bcl-2 overexpression in mammalian cells correlates with increased levels of the CycE/Cdk2 inhibitor p27 (Brady, 1996; Linette, 1996), hyperphosphorylated and inactive retinoblastoma (RB) tumor suppressor (Mazel, 1996), and increased levels of RB-related protein p130 (Lind, 1999; Vairo, 2000). The inhibitory effect of Bcl-2 on the cell cycle is independent of p53, the cdk4/6 inhibitor p16 and RB, but requires p130 and p27 (OíReilly, 1996; Vairo, 1996, 2000). The cell cycle inhibitory function of Bcl-2 can be separated from its cell survival function since the tyrosine residue, Y28, in the N-terminal BH4 domain is important for Bcl-2 inhibition of cell cycle re-entry, but is not required for cell survival (Huang, 1997). Although Buffy does not have an obvious BH4 domain, the cell cycle inhibitory function has been conserved between Buffy and Bcl-2. Thus, it will now be important to determine whether Buffy uses a mechanism similar to Bcl-2 to inhibit cell cycle progression (Quinn, 2003 and references therein).

GENE STRUCTURE

cDNA clone length - 1145 bp

Bases in 5' UTR - 157

Exons - 5

Bases in 3' UTR - 202

PROTEIN STRUCTURE

Amino Acids - 313

Structural Domains

Buffy encodes a protein that is 19% identical and 56% similar to human Bcl-2 over a 239 amino acid region and shares several conserved motifs with mammalian Bcl-2, including the BH1, BH2 and BH3 domains, and a C-terminal hydrophobic membrane anchor. Although an N-terminal BH4 domain present in pro-survival Bcl-2 proteins is not obvious in the Buffy sequence, there are two alpha-helical domains in the N-terminal region that might be functionally similar to the BH4 domain (Quinn, 2003).

EVOLUTIONARY HOMOLOGS

Information about anti-apoptotic BCL2 homologs can be found at death executioner Bcl-2 homologue (debcl), Evolutionary Homologs section.

date revised: 28 December 2003

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