Abstract
The calcium binding protein ALG-2 is upregulated in several types of cancerous tissues and cancer cell death may be a consequence of ALG-2 downregulation. Novel research suggests that ALG-2 is involved in membrane repair mechanisms, in line with several published studies linking ALG-2 to processes of membrane remodeling and transport, which may contribute to the fitness of cells or protect them from damage. To investigate the involvement of ALG-2 in cell recovery after membrane damage we disrupted the PDCD6 gene encoding the ALG-2 protein in DT-40 cells and exposed them to electroporation. ALG-2 knock-out cells were more sensitive to electroporation as compared to wild type cells. This phenotype could be reversed by reestablishing ALG-2 expression confirming that ALG-2 plays an important role in cell recovery after plasma membrane damage. We found that overexpression of wild type ALG-2 but not a mutated form unable to bind Ca2+ partially protected HeLa cells from digitonin-induced cell death. Further, we were able to inhibit the cell protective function of ALG-2 after digitonin treatment by adding a peptide with the ALG-2 binding sequence of ALIX, which has been proposed to serve as the ALG-2 downstream target in a number of processes including cell membrane repair. Our results suggest that ALG-2 may serve as a novel therapeutic target in combination with membrane damaging interventions.
Highlights
The EF-hand Ca2+-binding protein ALG-2 has been implicated in a variety of cellular processes including apoptosis, proliferation and protein trafficking among others
ALG-2 incapable of Ca2+ binding due to point mutations within EF-hand 1 and EF-hand 3, decreased cell viability compared to the EGFP control (~25% decrease after 1 hour and 27% after 3 hours). These findings indicate that ALG-2 may protect cells from death caused by plasma membrane damage and that the ALG-2 protective function is dependent on the propensity of ALG-2 to bind Ca2+
We present data providing evidence for the function of ALG-2 in cell survival after cell membrane damage induced by electroporation and digitonin treatment based on the following main findings: (1) an engineered chicken B cell line lacking the PDCD6 gene showed higher sensitivity to membrane damage by electroporation compared to the wild type cells and this phenotype could be reversed by reestablishing ALG-2 expression; (2) overexpression of ALG-2 in human cancer cells led to increased recovery after membrane damage caused by digitonin treatment
Summary
The EF-hand Ca2+-binding protein ALG-2 has been implicated in a variety of cellular processes including apoptosis, proliferation and protein trafficking among others (reviewed in [1,2]). ALG-2 was considered a proapoptotic protein based on its discovery as a mediator of T-cell apoptosis [3]. Whereas no direct mechanistic role for ALG-2 in cancer cell viability has been identified, recent discoveries have linked ALG-2 to membrane vesicle traffic and cargo packaging via its interaction with Sec31A [11,12]. It has been found to be associated with components of ESCRT important for a plethora of cellular processes associated with membrane remodeling, including endosome formation, fusion of autophagosomes and amphisomes with lysosomes as well as retrovirus budding among others (reviewed in [15])
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