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Application of flow cytometry to determine differential redistribution of cytochrome c and Smac/DIABLO from mitochondria during cell death signaling.

ABSTRACT: Mitochondrially mediated apoptosis is characterized by redistribution of proteins from mitochondria to cytoplasm following permeabilization of the outer mitochondrial membrane. We applied flow cytometry to quantify simultaneously the redistribution of two apoptogenic proteins, cytochrome c (cyt c) and Smac/DIABLO (Smac). Mammalian cells were treated with digitonin that selectively permeabilizes the plasma membrane. Following fixation, treated cells were infused successively with primary and secondary antibodies (the latter fluorescently tagged) enabling independent detection of cyt c and Smac. Digitonin-treated cells that retain cyt c or Smac in mitochondria generate strong fluorescence signals in flow cytometry. Cells in which cyt c or Smac have transited the outer mitochondrial membrane show greatly reduced fluorescence because the proteins are lost from the digitonin-permeabilized cells. Quantitative flow cytometry revealed that in 143B TK(-) cells treated with staurosporine, cyt c and Smac exit mitochondria asymmetrically, with cyt c redistribution preceding that of Smac. However, in HeLa cells likewise treated, cyt c and Smac exit mitochondria concurrently. Under other conditions of apoptotic induction, for example, 143B TK(-) cells treated with MT-21 (an apoptotic inducer that binds to the mitochondrial adenine nucleotide transporter), redistribution of Smac precedes that of cyt c. The various patterns of redistribution of these proteins were confirmed by immunocytochemical analysis and confocal microscopy. We conclude that flow cytometry can be employed effectively to quantify simultaneously the redistribution of cyt c and Smac from mitochondria to the cytosol. Moreover, differential redistribution of cyt c and Smac occurs under various conditions, thereby reflecting constraints on availability of these proteins to exit mitochondria after permeabilization of the outer membrane.

SUBMITTER: Ng H

PROVIDER: S-EPMC3407092 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:SMAC/Diablo, a pro-apoptotic protein, yet it is overexpressed in several cancer types. We have described a noncanonical function for SMAC/Diablo as a regulator of lipid synthesis during cancer cell proliferation and development. Here, we explore the molecular mechanism through which SMAC/Diablo regulates phospholipid synthesis. We showed that SMAC/Diablo directly interacts with mitochondrial phosphatidylserine decarboxylase (PSD) and inhibits its catalytic activity during synthesis of phosphatidylethanolamine (PE) from phosphatidylserine (PS). Unlike other phospholipids (PLs), PE is synthesized not only in the endoplasmic reticulum but also in mitochondria. As a result, PSD activity and mitochondrial PE levels were increased in the mitochondria of SMAC/Diablo-deficient cancer cells, with the total amount of cellular PLs and phosphatidylcholine (PC) being lower as compared to SMAC-expressing cancer cells. Moreover, in the absence of SMAC/Diablo, PSD inhibited cancer cell proliferation by catalysing the overproduction of mitochondrial PE and depleting the cellular levels of PC, PE and PS. Additionally, we demonstrated that both SMAC/Diablo and PSD colocalization in the nucleus resulted in increased levels of nuclear PE, that acts as a signalling molecule in regulating several nuclear activities. By using a peptide array composed of 768-peptides derived from 11 SMAC-interacting proteins, we identified six nuclear proteins ARNT, BIRC2, MAML2, NR4A1, BIRC5 and HTRA2 Five of them also interacted with PSD through motifs that are not involved in SMAC binding. Synthetic peptides carrying the PSD-interacting motifs of these proteins could bind purified PSD and inhibit the PSD catalytic activity. When targeted specifically to the mitochondria or the nucleus, these synthetic peptides inhibited cancer cell proliferation. To our knowledge, these are the first reported inhibitors of PSD acting also as inhibitors of cancer cell proliferation. Altogether, we demonstrated that phospholipid metabolism and PE synthesis regulated by the SMAC-PSD interaction are essential for cancer cell proliferation and may be potentially targeted for treating cancer.

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Application of flow cytometry to determine differential redistribution of cytochrome c and Smac/DIABLO from mitochondria during cell death signaling.

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