Erythrocyte survival is promoted by plasma and suppressed by a Bak-derived BH3 peptide that interacts with membrane-associated Bcl-XL

Abstract

Erythrocytes have a defined lifespan in vivo, and the signals that maintain their survival in circulation or trigger their death are unknown. Here, we investigated the control of erythrocyte survival and death in an in vitro culture system where erythrocytes survived for 10 days in serumfree medium in the presence or absence of bovine serum. Death of the cells in culture was correlated with increased exposure of phosphatidylserine and increased levels of intracellular calcium. Cell death could be suppressed by supplementing the medium with human plasma or serum, resulting in a doubling of the lifespan to 20 days. Freshly isolated erythrocytes and cultured erythrocytes were both found to express Bcl-X L and, to a lesser extent, Bak in membrane protein extracts. Treatment of the cells with a Bak-derived BH3 peptide fused to the internalization sequence of the antennapedia protein, which has previously been shown to enter cells by diffusion and antagonize Bcl-X L , resulted in substantial cell death in erythrocyte cultures. BH3-induced death was accompanied by an immediate increase in accumulation of intracellular calcium and could be suppressed by plasma, but not by the caspase inhibitor zVAD. A BH3 peptide mutated at amino acid 78 of full-length Bak required for heterodimerization with Bcl-X L had no effect on cell viability or calcium levels. We conclude that the BH3 peptide accelerates erythrocyte death through antagonization of Bcl-X L . The data suggest that erythrocyte survival is promoted by survival factors in plasma and by membrane- IntroductionCell death and survival in hematopoietic cells is regulated by signals from cell-surface receptors that either promote apoptosis, such as Fas and tumor necrosis factor (TNF), or repress cell death, such as receptors for cytokines and survival factors such as erythropoietin (EPO), interleukin 3, and insulinlike growth factor I (IGF-I). [1][2][3] Ligation of the receptors for Fas/TNF can lead directly to activation of caspases, which can act as effectors of cell death by catalyzing structural changes associated with apoptosis, including cytoskeletal reorganization and DNA fragmentation. Survival factors mediate signals through several kinases, such as phosphoinositol 3 kinase and AKT, ultimately leading to transcriptional events or to regulation of Bcl-2 family proteins. 4 The Bcl-2 family acts as central regulators of cell death in many cells [5][6][7] and is divided into 2 main groups consisting of suppressors of apoptosis, including Bcl-2, Bcl-X L , Bcl-w, and Mcl-1, and potent apoptosis promoters, including Bax, Bak, Bad, and Bid. Bcl-2 and Bcl-X L survival activity can be antagonized by heterodimerization with proteins containing a BH3 domain, which is the essential domain for the binding and proapoptotic activity of Bak, Bax, Bik, and Bid. 8 Bcl-2 and Bcl-X L are expressed in outer mitochondrial membranes where antagonization of their function is associated with rupture of the membrane, release of the proapoptotic factors cytochrome c, and activation of caspas...

Survival and Senescence of Human Young Red Cellsin Vitro

“…In partial agreement with observations by Walsh et al [21], who studied whole red cell population under the same culture conditions, we found that a lower percentage of young, middle aged and old cells could be recovered from cultures in plasma-free medium than from those with plasma. Furthermore, neocytes showed the lowest survival rate at each time point in both culture mediums ( p <0,01) (Fig.…”

Ankyrin Peptide Blocks Falcipain-2-mediated Malaria Parasite Release from Red Blood Cells

“…To determine the effect of AnkP peptide on the growth and development of P. falciparum, we utilized a recent technique where peptides were delivered into intact RBCs by fusion with the Antennapedia homeoprotein internalization domain (22). Human erythrocytes infected with stage-specific parasites were incubated with 100 -250 M of the fused peptide (AntAnkP).…”

“…Peptides were synthesized by fusing with the 16 amino acid internalization sequence of Antennapedia (Ant) homeoprotein, which permits the uptake of peptides into cells via facilitated diffusion (21,22). Human erythrocytes infected with stage-specific P. falciparum were cultured in the presence of Ant-AnkP peptide, the Ant peptide alone, AnkP peptide alone, or a mutant form of the Ant-AnkP peptide where the AnkP portion was scrambled to eliminate the signature sequence of the cleavage site ( Table I).…”

Mitochondria in hematopoiesis and hematological diseases

“…Altogether, these results suggest that the mitochondrial pathway to caspase activation is tightly regulated along the erythroid pathway of differentiation, a regulation that involves Bcl-X L and probably other Bcl-2-related proteins at the mitochondrion level (Aerbajinai et al, 2003). Surprisingly enough, a membrane-associated Bcl-X L was reported also to protect mature erythrocytes, which lack mitochondria, from death (Walsh et al, 2002).…”